CD16A binding proteins and use for the treatment of immune disorders

ABSTRACT

CD16A binding proteins useful for the reduction of a deleterious immune response are described. In one aspect, humanized anti-cd16A antibodies, optionally lacking effector function, are used for the treatment of immune disorders such as idiopathic thrombocytopenic purpura and autoimmune hemolytic anemia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/449,566, filed May 29, 2003, which is now U.S. Pat. No. 7,351,803,and which claims benefit of provisional patent application No.60/384,689, filed May 30, 2002, and provisional patent application No.60/439,320, filed Jan. 10, 2003, the entire contents of each of whichare incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

The invention relates to CD16A binding proteins and methods fortreatment of immune disorders. The invention finds application in thefields of biomedicine and immunology.

BACKGROUND

Fcγ receptors (FcγR) are cell surface receptors that bind the Fc regionof immunoglobulin G (IgG) molecules. Among other functions, thesereceptors couple the formation of antibody-antigen complexes to effectorcell responses. For example, cross-linking of activating Fcγ receptorsby immune complexes can result in the phagocytosis of pathogens, killingof foreign and transformed cells by-direct cytotoxicity, the clearanceof toxic substances, and the initiation of an inflammatory response.Notably, the Fcγ receptors play a key role in autoimmunity. Autoantibodybinding to activating Fc receptors triggers the pathogenic sequalae ofautoimmune diseases such as idiopathic thrombocytopenic purpura,arthritis, systemic lupus erythrematosus, autoimmune hemolytic anemia,and others.

In humans and rodents there are three classes of Fcγ receptors,designated FcγRI, FcγRII, and FcγRIII (see, Ravetch and Bolland, 2001Annual Rev. Immunol 19:275-90; and Ravetch and Kinet, 1991, Annual Rev.Immunol. 9:457-92). FcγRI sites are generally occupied by monomeric IgG,while RII and RIII receptors are generally unoccupied and available tointeract with immune complexes. FcγRI, also called CD64, binds monomericIgG with high affinity, and is present on monocytes and macrophages.FcγRII, also called CD32, binds to multimeric IgG (immune complexes oraggregated IgG) with moderate affinity, and is present on a variety ofcell types, including B cells, platelets, neutrophils, macrophages andmonocytes. FcγRIII, also called CD16, binds to multimeric IgG withmoderate affinity and is the predominant activating FcγR on myeloidcells. FcγRIII is found in two forms. FcγRIIIA (CD16A), a transmembranesignaling form (50-65 kDa), is expressed by NK cells, monocytes,macrophages, and certain T cells. FcγRIIIB (CD16B), aglycosyl-phosphatidyl-inositol anchored form (48 kDa) form, is expressedby human neutrophils. See, e.g., Scallon et al., 1989, Proc. Natl. Acad.Sci. U.S.A. 86:5079-83 and Ravetch et al., 1989, J. Exp. Med.170:481-97. Protein and nucleic acid sequences for CD16A are reported inGenbank as accession numbers P08637 (protein) and X52645 (nucleic acid)and in SWISS-PROT as accession number CAA36870. Protein and nucleic acidsequences for CD16B are reported in Genbank as accession numbers O75015(protein) and X16863 (nucleic acid) and in SWISS-PROT as CAA34753.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a CD16A binding protein that maybe used for treatment of an individual with an autoimmune disease. CD16Abinding proteins of the invention are other than mouse antibodies, andinclude chimeric, human and humanized anti-CD16A monoclonal antibodies,fragments thereof, single chain antibodies, and other binding proteinscomprising a V_(H) domain and/or a V_(L) domain.

In one aspect the CD16A binding protein comprises a Fc region derivedfrom a human IgG heavy chain (e.g., a Fc region derived from human IgG₁)where the Fc region lacks effector function and/or is modified to reducebinding to a Fc effector ligand. In one embodiment, the CD16A bindingprotein is not glycosylated, for example, due to a substitution atresidue 297 of the Fc region.

In one aspect, the CD16A binding protein is a humanized 3G8 antibodywith a V_(H) domain comprising three complementarity determining regions(CDRs) derived from the V_(H) domain of mouse monoclonal antibody 3G8.In one embodiment, the V_(H) domain has the sequence of the V_(H) domainof Hu3G8VH-1. In one embodiment, the CDRs of the binding protein havethe sequence of the mouse CDRs. In some versions, the V_(H) domain CDRsdiffer from those of 3G8 at least by one or more of the followingsubstitutions: Val at position 34 in CDR1, Leu at position 50 in CDR2,Phe at position 52 in CDR2, Asn at position 54 in CDR2, Ser at position60 in CDR2, Ser at position 62 in CDR2, Tyr at position 99 in CDR3, andAsp at position 101 of CDR3. In one embodiment, the V_(H) domain has thesequence of the V_(H) domain of Hu3G8V_(H)-22. In one embodiment V_(H)domain comprises an FR3 domain having the sequence of SEQ ID NO:51. TheV_(H) domain may be linked to an antibody heavy chain constant domain,for example the human Cγ1 constant domain.

In some versions the CD16A binding protein has a V_(H) domain having asequence set forth in Table 4. In some versions the CD16A bindingprotein has a V_(H) domain that differs from the sequence of Hu3G8VH-1by one or more of the substitutions shown in Table 1.

In one aspect, the CD16A binding protein is a humanized 3G8 antibodywith a V_(L) domain comprising three complementarity determining regions(CDRs) derived from the V_(L) domain of mouse monoclonal antibody 3G8.In one embodiment, the CDRs of the binding protein have the sequence ofthe mouse CDRs. In some versions, the V_(L) domain CDRs differ fromthose of 3G8 at least by one or more of the following substitutions: Argat position 24 in CDR1; Ser at position 25 in CDR1; Tyr at position 32in CDR1; Leu at position 33 in CDR1; Ala at position 34 in CDR1; Asp,Trp or Ser at position 50 in CDR2; Ala at position 51 in CDR2; Ser atposition 53 in CDR2; Ala or Gln at position 55 in CDR2; Thr at position56 in CDR2; Tyr at position 92 in CDR3; Ser at position 93 in CDR3; andThr at position 94 in CDR3. In one embodiment, the V_(L) domain has thesequence of the V_(•L) domain of Hu3G8VL-1, Hu3G8VL-22 or Hu3G8VL-43.The V_(L) domain may be linked to an antibody light chain constantdomain, for example the human Cκ constant region.

In some versions the CD16A binding protein has a V_(L) domain having asequence set forth in Table 5. In some versions the CD16A bindingprotein has a V_(L) domain that differs from the sequence of Hu3G8VL-1by one or more of the substitutions shown in Table 2.

In one aspect, the CD16A binding protein comprises both a V_(H) domainand a V_(L) domain, as described above (which may be prepared bycoexpression of polynucleotides encoding heavy and light chains).Optionally the humanized heavy chain variable region comprises asequence set forth in Table 4 and/or the a humanized light chainvariable region comprises a sequence set forth in Table 5. For example,in exemplary embodiments, the binding protein has a heavy chain variableregion having the sequence of SEQ ID NO:113 and a light chain variableregion having the sequence of SEQ ID NO:96, 100 or 1118. In anotherexemplary embodiment, the binding protein has a heavy chain variableregion having the sequence of SEQ ID NO:109 and light chain variableregions having the sequence of SEQ ID NO:96. In another exemplaryembodiment, the binding protein has a heavy chain variable region havingthe sequence of SEQ ID NO:104 and light chain variable regions havingthe sequence of SEQ ID NO:96.

In an embodiment, the CD16A binding protein is tetrameric antibodycomprising two light chains and two heavy chains, said light chainscomprising a V_(L) domain and a light chain constant domain and saidheavy chains comprising a V_(H) domain and a heavy chain constantdomain. In an embodiment, the light chain constant domain is human Cκand/or the heavy chain constant region is Cγ1.

In one embodiment of the invention, the CD16A binding protein comprisesan antigen binding site that binds CD16A or sCD16A with a bindingconstant of less than 5 nM.

In one embodiment, the CD16A binding protein comprises an aglycosyl Fcregion that has reduced binding to at least one Fc effector ligandcompared to a reference CD16A binding protein that comprises anunmodified Fc region (e.g., a human IgG₁ Fc domain glycosylated atposition 297). The Fc effector ligand can be Fcy RIII or the C1qcomponent of complement.

In one embodiment, the invention provides a CD16A binding protein thatis humanized antibody that binds to CD16A and inhibits the binding of Fcto CD16.

In an aspect, the invention provides a pharmaceutical compositioncomprising of CD16A binding protein described herein and apharmaceutically acceptable excipient.

In an aspect, the invention provides an isolated polynucleotide,optionally an expression vector, encoding a V_(H) domain of a CD16Abinding protein described herein. In an aspect, the invention providesan isolated nucleic acid, optionally an expression vector, encoding aV_(L) domain of a CD16A binding protein described herein. In an aspect,the invention provides a cell, optionally a mammalian cell, comprising apolynucleotide described herein. In an aspect, the invention a cellline, optionally a mammalian cell line, expressing a CD16A bindingprotein described herein.

The invention further provides a method of reducing an deleteriousimmune response (or undesired immune response) in a mammal comprisingadministering to a mammal a CD16A binding protein described herein. Inan embodiment, reducing the deleterious immune response comprisesprotecting against antibody-mediated platelet depletion.

In one aspect, the invention provides a method of treating andeleterious immune response in a mammal without inducing neutropenia inthe mammal (e.g., severe neutropenia or moderate neutropenia), where themethod comprises administering to the mammal a CD16A binding proteinhaving an Fc region derived from human IgG, and where the amino acid atposition 297 of the Fc region is aglycosyl.

In embodiments of the above-described methods, the deleterious immuneresponse is an inflammatory response, for example, an inflammatoryresponse caused by an autoimmune disease. In an embodiment, theinflammatory response is caused by idiopathic thrombocytopenic purpura(ITP), rheumatoid arthritis (RA), systemic lupus erythrematosus (SLE),autoimmune hemolytic anemia (AHA), scleroderma, autoantibody triggeredurticaria, pemphigus, vasculitic syndromes, systemic vasculitis,Goodpasture's syndrome, multiple sclerosis (MS), psoriatic arthritis,ankylosing spondylitis, Sjogren's syndrome, Reiter's syndrome,Kowasaki's disease, polymyositis and dermatomyositis. Other examples ofdiseases or conditions that can be treated according to the inventionalso include any diseases susceptible to treatment with intravenousimmunoglobulin (IVIG) therapy (e.g., allergic asthma). The inventionprovides CD16A binding proteins that both protect against autoimmunediseases and do not result in significant neutrophil diminution in amammal. In an embodiment, the CD16A binding proteins are anti-CD16Aantibodies. These CD16A binding proteins are particularly advantageousfor use as human therapeutics. In one aspect, the invention provides amethod of treating an autoimmune disease in a mammal without neutrophildiminution or neutropenia in the mammal, by administering a CD16Abinding protein having an Fc region derived from human IgG and anaglycosyl amino acid at position 297 of each of the C_(H)2 domains ofthe Fc region.

In yet another aspect, the invention provides a method of inhibiting thebinding of IgG antibodies to FcγRIII on a cell by contacting the cellwith a CD16A binding protein under conditions in which the CD16A bindingprotein binds the FcγRIII on the cell.

In one aspect, the invention provides a method of making a CD16A bindingprotein with improved therapeutic efficacy in treating an deleteriousimmune response, comprising the following steps: i) obtaining a firstCD16A binding protein, where the first CD16A binding protein comprisesan Fc region derived from IgG; and ii) modifying the Fc region of thefirst CD16A binding protein to produce a second CD16A binding proteinthat is aglycosylated at position 297 of the Fc region, where the secondCD16A binding protein is more effective in treating the deleteriousimmune response when administered to a mammal than the first CD16Abinding protein.

In one aspect, the invention provides a method of making a CD16A bindingprotein with improved therapeutic efficacy in treating an deleteriousimmune response, comprising the following steps: i) obtaining a firstCD16A binding protein, wherein the first CD16A binding protein comprisesan Fc region derived from IgG; and ii) modifying the Fc region of thefirst CD16A binding protein to produce a second CD16A binding proteinthat has reduced binding to an Fc effector ligand compared to theunmodified Fc region of the first CD16A binding protein, where thesecond CD16A binding protein is more effective in treating thedeleterious immune response when administered to a mammal than the firstCD16A binding protein. In one embodiment, the Fc effector ligand isFcγRIII or the C1q component of complement.

In one aspect the method involves administering a CD16A binding proteinto reduce an deleterious immune response in a subject without elicitingone or more significant deleterious effects that result from 3G8administration, or eliciting significantly lower levels of such effectsthan does administration of murine 3G8.

In one embodiment of the invention, the improved therapeutic efficacy intreating a deleterious immune response comprises improved effectivenessat protecting against antibody-mediated platelet depletion. Thedeleterious immune response is optionally due to idiopathicthrombocytopenic purpura (ITP) or the administration of murinemonoclonal antibody 6A6 to a muFcγRIII−/−, huFcγRIIIA transgenic mouse.

The invention provides the use of a CD16A binding protein comprising anFc region derived from a human IgG heavy chain, wherein the Fc regionlacks effector function, for treatment of an immune disorder or forpreparation of a medicament for treatment of an immune disorder.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows results from an ELISA for binding of sCD16A by CD16Abinding proteins. Hu3G8-24.43 is an antibody with the heavy chainHu3G8VH-24, and the light chain Hu3G8VL-43. Hu3G8-5.1 is an antibodywith the heavy chain Hu3G8VH-5, and the light chain Hu3G8VL-1. Ch3G8 isthe chimeric 3G8 antibody. Hu1gG1 is an irrelevant immunoglobulin.

FIG. 2 shows results of an assay for binding of humanized and chimericantibodies to CHO-K1 cells expressing the extracellular domain of CD16A.Hu3G8-22.1 is an antibody with the heavy chain Hu3G8VH-22, and the lightchain Hu3G8VL-1. Hu3G8-5.1 is an antibody with the heavy chainHu3G8VH-5, and the light chain Hu3G8VL-1. Hu3G8-22.43 is an antibodywith the heavy chain Hu3G8VH-22, and the light chain Hu3G8VL-43. N297Qindicates the antibody is aglycosylated.

FIG. 3 shows results of a cell based competition assay. Theaglycosylated humanized antibodies shown compete with aglycosylatedchimeric antibody for binding to CHO-K1 cells expressing theextracellular domain of CD16A.

FIG. 4 shows inhibition of binding of sCD16A to immune complexes.Hu3G8-1.1 is an antibody with the heavy chain Hu3G8VH-1, and the lightchain Hu3G8VL-1.

FIG. 5 shows ITP protection in mice injected i.v. with mAb 3G8 (0.5μg/g) or human IVIG (1 mg/g) one hour before ch6A6 i.p injection.

FIG. 6 shows ITP protection in mice injected i.v. with mAb 3G8 (0.5μg/g) or human IVIG (1 mg/g) one hour before ch6A6 i.v injection.

FIG. 7 shows the absence of ITP protection in mice injected i.v. withch3G8 (0.5 μg/g) one hour before 6A6 i.p. injection.

FIG. 8 shows protection from ITP in mice injected i.v. with ch3G8 N297Qone hour before ch6A6 i.p injection.

FIG. 9 shows protection from ITP in mice injected i.v. with ch3G8 N297Qone hour before ch6A6 i.v injection.

FIG. 10 shows the results of FACS scans of neutrophils followingadministration of CD16A binding protein or controls. The x-axis showslabeling with antibody to CD16, and the y-axis shows labeling withantibody to the Gr-1 antigen. The upper right quadrant showsneutrophils; the upper left quadrant shows other granulocytes andneutrophils that no longer stain with 3G8-FITC.

FIG. 11 shows prevention of AIHA with a humanized anti-CD16 antibody.

FIG. 12 shows inhibition of ch4D5 mediated ADCC by humanized 3G8antibodies.

FIG. 13 shows inhibition of ch4-4-20 mediated ADCC by mouse 3G8 (FIG.13A) and humanized 3G8 antibodies (FIG. 13B).

FIG. 14 shows protection of FcγRIII−/−, hCD16A, hCD32A mice against ITPby administration of hu3G8-5.1.

FIG. 15 shows protection of FcγRIII−/−, hCD16A mice against ITP byadministration of hu3G8-5.1 N297Q. FIG. 15(A) shows data points for eachdose at indicated times. FIG. 15(B) shows dose response at the 5 hourtime point.

FIG. 16 shows the therapeutic effect of administration of aglycosylatedhumanized antibody subsequent to mice in which thrombocytopenia has beeninduced. FIG. 16(A) shows administration of Hu3G8-5.1-N297Q. FIG. 16(B)shows administration of Hu3G8-22.1-N297Q and Hu3G8-22.43-N297Q.

FIG. 17 shows the therapeutic effect of a humanized anti-CD16A antibodyin treatment of autoimmune hemolytic anemia.

DETAILED DESCRIPTION 1. Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. The practice of the present invention willemploy, unless otherwise indicated, conventional techniques of molecularbiology (including recombinant techniques), microbiology, cell biology,biochemistry, nucleic acid chemistry, and immunology, which are withinthe skill of the art. Such techniques are explained fully in theliterature, such as, Current Protocols in Immunology (J. E. Coligan etal., eds., 1999, including supplements through 2001); Current Protocolsin Molecular Biology (F. M. Ausubel et al., eds., 1987, includingsupplements through 2001); Molecular Cloning: A Laboratory Manual, thirdedition (Sambrook and Russel, 2001); PCR: The Polymerase Chain Reaction,(Mullis et al., eds., 1994); The Immunoassay Handbook (D. Wild, ed.,Stockton Press NY, 1994); Bioconjugate Techniques (Greg T. Hermanson,ed., Academic Press, 1996); Methods of Immunological Analysis (R.Masseyeff, W. H. Albert, and N. A. Staines, eds., Weinheim: VCH Verlagsgesellschaft mbH, 1993), Harlow and Lane Using Antibodies: A LaboratoryManual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1999; and Beaucage et al. eds., Current Protocols in Nucleic AcidChemistry John Wiley & Sons, Inc., New York, 2000).

The terms “heavy chain,” “light chain,” “variable region,” “frameworkregion,” “constant domain,” and the like, have their ordinary meaning inthe immunology art and refer to domains in naturally occurringimmunoglobulins and the corresponding domains of synthetic (e.g.,recombinant) binding proteins (e.g., humanized antibodies). The basicstructural unit of naturally occurring immunoglobulins (e.g., IgG) is atetramer having two light chains and two heavy chains. Usually naturallyoccurring immunoglobulin is expressed as a glycoprotein of about 150,000daltons, although, as described below, IgG can also be produced in anonglycosylated form. The amino-terminal (“N”) portion of each chainincludes a variable region of about 100 to 110 or more amino acidsprimarily responsible for antigen recognition. The carboxy-terminal(“C”) portion of each chain defines a constant region, with light chainshaving a single constant domain and heavy chains usually having threeconstant domains and a hinge region. Thus, the structure of the lightchains of an IgG molecule is N-V_(L)-C_(L)-C and the structure of IgGheavy chains is N-V_(H)-C_(H)1-H-C_(H)2-C_(H)3-C (where H is the hingeregion). The variable regions of an IgG molecule consists of thecomplementarity determining regions (CDRs), which contain the residuesin contact with antigen and non-CDR segments, referred to as frameworksegments, which maintain the structure and determine the positioning ofthe CDR loops. Thus, the V_(L) and V_(H) domains have the structureN-FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4-C.

As used herein, the terms “CD16A binding protein,” “CD16A antibody,” and“anti-CD16A antibody,” are used interchangeably and refer to a varietyof immunoglobulin-like or immunoglobulin-derived proteins. “CD16Abinding proteins” bind CD16A via an interaction with V_(L) and/or V_(H)domains (as distinct from Fc-mediated binding). Examples of CD16Abinding proteins includes chimeric, humanized and human antibodies(e.g., comprising 2 heavy and 2 light chains), fragments thereof (e.g.,Fab, Fab′, F(ab′)₂, and Fv fragments), bifunctional or multifunctionalantibodies (see, e.g., Lanzavecchia et al., 1987, Eur. J. Immunol.17:105), single chain antibodies (see, e.g., Bird et al., 1988, Science242:423-26), fusion proteins (e.g., phage display fusion proteins),“minibodies” (see, e.g., U.S. Pat. No. 5,837,821) and other antigenbinding proteins comprising a V_(L) and/or V_(H) domain or fragmentthereof. In one aspect, the CD16A binding protein is a “tetramericantibody” i.e., having generally the structure of a naturally occurringIgG and comprising both variable and constant domains, (i.e., two lightchains comprising a V_(L) domain and a light chain constant domain, suchas human Cκ and two heavy chains comprising a V_(H) domain and a heavychain hinge and constant domains, such as human Cγ1). Except asexpressly noted, the mouse antibody 3G8 is specifically excluded fromthe definition of CD16A binding protein.

When referring to binding proteins or antibodies (as broadly definedherein) the assignment of amino acids to each domain is in accordancewith the definitions of Kabat, SEQUENCES OF PROTEINS OF IMMUNOLOGICALINTEREST (National Institutes of Health, Bethesda, Md., 1987 and 1991).Amino acids from the variable regions of the mature heavy and lightchains of immunoglobulins are designated by the position of an aminoacid in the chain. Kabat described numerous amino acid sequences forantibodies, identified an amino acid consensus sequence for eachsubgroup, and assigned a residue number to each amino acid. Kabat'snumbering scheme is extendible to antibodies not included in hiscompendium by aligning the antibody in question with one of theconsensus sequences in Kabat by reference to conserved amino acids. Thismethod for assigning residue numbers has become standard in the fieldand readily identifies amino acids at equivalent positions in differentantibodies, including chimeric or humanized variants. For example, anamino acid at position 50 of a human antibody light chain occupies theequivalent position to an amino acid at position 50 of a mouse antibodylight chain. Thus, as used herein in the context of chimeric orhumanized antibodies, a reference such as “at position 297 of the Fcregion” refers to the amino acid position in an immunoglobulin chain,region of an a immunoglobulin chain, or region of a polypeptide derivedfrom an immunoglobulin chain, that corresponds to position 297 of thecorresponding human immunoglobulin.

The “Fc region” of immunoglobulins refers to the C-terminal region of animmunoglobulin heavy chain. Although the boundaries of the Fc region mayvary somewhat, usually the Fc region is from about position 226-230extending to the carboxy terminus of the polypeptide (and encompassingthe C_(H)2 and C_(H)3 domains). Sequences of human Fc regions are foundin Kabat, supra. In addition, a variety of allotypic variants are knownto exist.

An “Fc effector ligand” is a ligand that binds to the Fc region of anIgG antibody, thereby activating effector mechanisms resulting in theclearance and destruction of pathogens. Fc effector ligands includethree cellular Fc receptors types—FcRγI, FcRγII, and FcRγIII. Themultiple isoforms of each of the three Fc receptor types are alsoincluded. Accordingly, the term “Fc effector ligand” includes bothFcRγIIIA (CD16A) and FcRγIIIB (CD16B). The term “Fc effector ligand”also includes the neonatal Fc receptor (Fcγn) and the C1q component ofcomplement. Binding of IgG to the Fc receptors triggers a variety ofbiological processes including antibody-dependent cell-mediatedcytotoxicity (ADCC), release of inflammatory mediators, control ofantibody production, clearance of immune complexes and destruction ofantibody-coated particles. Binding of the C1q component of complement toIgG activates the complement system. Activation of complement playsimportant roles in opsonization, lysis of cell pathogens, andinflammatory responses.

As used herein, an Fc region that “lacks effector function” does notbind the Fc receptor and/or does not bind the C1q component ofcomplement and trigger the biological responses characteristic of suchbinding.

The term “glycosylation site” refers to an amino acid residue that isrecognized by a mammalian cell as a location for the attachment of sugarresidues. Amino acid residues to which carbohydrates, such asoligosaccharides, are attached are usually asparagine (N-linkage),serine (O-linkage), and threonine (O-linkage) residues. The specificsites of attachment usually have a characteristic sequence of aminoacids, referred to as a “glycosylation site sequence.” The glycosylationsite sequence for N-linked glycosylation is: -Asn-X-Ser- or -Asn-X-Thr-,where X can be any of the conventional amino acids, other than proline.The Fc region of human IgG has two glycosylation sites, one in each ofthe C_(H)2 domains. The glycosylation that occurs at the glycosylationsite in the C_(H)2 domain of human IgG is N-linked glycosylation at theasparagine at position 297 (Asn 297).

The term “chimeric,” when referring to antibodies, has the ordinarymeaning in the art and refers to an antibody in which a portion of aheavy and/or light chain is identical to or homologous with an antibodyfrom one species (e.g., mouse) while the remaining portion is identicalto or homologous with an antibody of another species (e.g., human).

As used herein, the term “humanized” has its usual meaning in the art.In general terms, humanization of a non-human antibody involvessubstituting the CDR sequences from non-human immunoglobulin V_(L) andV_(H) regions into human framework regions. Further, as used herein,“humanized” antibodies may comprise additional substitutions andmutations in the CDR and/or framework regions introduced to increaseaffinity or for other purposes. For example, substitution of nonhumanframework residues in the human sequence can increase affinity. See,e.g., Jones et al., 1986, Nature 321:522-25; Queen et al., 1989, Proc.Natl. Acad. Sci. U.S.A. 86:10029-33; Foote and Winter, 1992, J. Mol.Biol. 224:487-99; Chothia et al., 1989, Nature 342:877-83; Riechmann etal., 1988, Nature 332:323-27; Co et al., 1991, Proc. Natl. Acad. Sci.U.S.A. 88:2869-73; Padlan, 1991, Mol. Immunol. 28:489-98. The resultingvariable domains have non-human CDR sequences and framework sequencesderived from human antibody framework sequence(s) or a human consensussequence (e.g., as disclosed in Kabat, supra). A variety of differenthuman framework regions may be used singly or in combination as a basisfor the humanized monoclonal antibodies of the present invention. Theframework sequences of a humanized antibody are “substantially human,”by which is meant that at least about 70% of the human antibodysequence, usually at least about 80% human, and most often at leastabout 90% of the framework sequence is from human antibody sequence. Insome embodiments, the substantially human framework comprises a serineat position 113 of the V_(H) FR4 domain (e.g., SEQ ID NO: 64). As usedherein, a “humanized antibody” includes, in addition to tetramericantibodies, single chain antibodies, antibody fragments and the likethat comprise CDRs derived from a non-human antibody and frameworksequences derived from human framework regions.

As used herein, “mammals” include humans, non-human primates, rodents,such as, mice and rats, and other mammals.

As used herein, “neutropenia” has its ordinary meaning, and refers to astate in which the number of neutrophils circulating in the blood isabnormally low. The normal level of neutrophils in human blood variesslightly by age and race. The average adult level is about 1500cells/mm³ of blood. Neutrophil counts less than 500 cells/mm³ result ingreat risk of severe infection. Generally, in humans, severe neutropeniais defined by a blood neutrophil count less than about 500 cells/mm³,and moderate neutropenia is characterized by a blood neutrophil countfrom about 500-1000 cells/mm³.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the disease course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Therapeutic effects of treatment includewithout limitation, preventing occurrence or recurrence of disease,alleviation of symptoms, diminishment of any direct or indirectpathological consequences of the disease, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis.

An “effective amount” is an amount sufficient to effect a beneficial ordesired clinical result upon treatment. An effective amount can beadministered to a patient in one or more doses. A “therapeuticallyeffective amount” is an amount that is sufficient to palliate,ameliorate, stabilize, reverse or slow the progression of the disease,or otherwise reduce the pathological consequences of the disease, orreduce the symptoms of the disease. The amelioration or reduction neednot be, and usually is not, permanent, but may be for a period of timeranging from at least one hour, at least one day, or at least on week ormore. The effective amount is generally determined by the physician on acase-by-case basis and is within the skill of one in the art. Severalfactors are typically taken into account when determining an appropriatedosage to achieve an effective amount. These factors include age, sexand weight of the patient, the condition being treated, the severity ofthe condition and the form and effective concentration of the bindingprotein administered. An “inflammation reducing amount” is an amountthat reduces inflammation in a subject. A reduction in inflammation canbe assessed by art known criteria, including decreased C-reactiveprotein levels, decreased consumption of complement, reduced immunecomplex deposition at sites of inflammation (e.g., joints in subjectswith RA, kidney in subjects with lupus, myelin sheath, etc.), reducedcytokine release, migration of macrophages and neutrophils, and thelike.

“Substantial sequence identity,” as used herein, refers to two or moresequences or subsequences (e.g., domains) that have at least about 80%amino acid residue identity, preferably at least about 90%, or at leastabout 95% identity when compared and aligned for maximum correspondence.Sequence identity between two similar sequences (e.g., antibody variableregions) can be measured by (1) aligning the sequences to maximize thetotal number of identities across the entire length of the sequences, oracross the entire length of the shorter of the two sequences, if ofdifferent lengths (and where the length of the aligned sequences orshorter of the aligned sequences is “L” residues); (2) counting thenumber of positions (not including the number “E” residues designated asexcluded from the comparison) at which there is an amino acid identity,where the number of identities is designated “N”; (3) and dividing the Nby the “L” minus “E.” For example, in a comparison of two sequences eachof length 80 residues, in which 6 specific residues are excluded fromthe comparison and for which there are 65 identities in the remaining 74positions, the sequence identity would be N/(L−E) or 65/(80−6) or 87.8%.(Residues might be specified as “excluded” from the calculation when,for illustration but not limitation, they are in a non-antibody domainof fusion protein.) Alternatively, optimal alignment and sequenceidentity can be calculated by computerized implementations of algorithmsdescribed in Smith & Waterman, 1981, Adv. Appl. Math. 2:482 [localhomology algorithm], Needleman & Wunsch, 1970, J. Mol. Biol. 48:443[homology alignment algorithm], Pearson & Lipman, 1988, Proc. Natl.Acad. Sci. USA 85:2444 [search for similarity method], or Altschul etal., 1990, J. Mol. Biol. 215:403-10 [BLAST algorithm]. See Ausubel etal., supra and GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.). When using any of the aforementioned algorithms, the defaultparameters (for Window length, gap penalty, etc.) are used. An aminoacid or nucleic acid sequence is “substantially similar to” a secondsequence when the degree of sequence identity is at least about 70%identical, preferably at least about 80%, or at least about 90%, or evenat least about 95%, identical. Sequences that are substantiallyidentical are also substantially similar.

As used herein, a polypeptide, polypeptide domain or region, or aminoacid sequence is “derived from” another when the two sequences areidentical or substantially similar and have a similar biologicalfunction. For example, in a humanized mouse monoclonal antibody thecomplementary determining regions (CDRs) are “derived from” thecorresponding CDRs of the mouse monoclonal antibody, and the variabledomain framework regions can be “derived from” framework sequences ofthe corresponding human antibody. It will be apparent that one domain,etc., can be derived from a parental domain, etc., even though the twodiffer in sequence due to, for example, the introduction of mutationsthat affect, or alternatively do not change, binding affinity or otherproperties of the protein in which the domain, etc., is contained, suchas those described herein. It will also be understood that normally adomain, etc., “derived from” a parental domain, etc., is made, producedor designed using materials (e.g. genetic material) or information(e.g., nucleotide or amino acid sequence) from the parental molecule.

Standard abbreviations are used for amino acids: alanine, Ala (A);serine, Ser (S); threonine, Thr (T); aspartic acid, Asp (D); glutamicacid, Glu (E); asparagine, Asn (N); glutamine, Gln (O); arginine, Arg(R); lysine, Lys (K); isoleucine, Ile (I); leucine, Leu (L); methionine,Met (M); valine, Val (V); phenylalanine, Phe (F); tyrosine, Tyr (Y);tryptophan, Trp (W); glycine, Gly (G); histidine, H is (H): proline, Pro(P); and cysteine, Cys (C).

2. Introduction

The FcγRIIIA receptor, CD16A, plays a role in coupling cytotoxic andimmune complex antibodies to effector responses. It is believed that theinteraction of the FcγRIIIA receptor and immunoglobulin aggregates (e.g.immune complexes) present in autoimmune diseases and other pathogenicconditions results in a deleterious inflammatory response in subjects.Without intending to be bound by a specific mechanism, it is believedthat reducing the interaction of the FcγRIIIA receptor (generallyreferred to herein as “CD16A” or “the CD16A receptor” and immunoglobulinaggregates will alleviate this inflammatory response. Also withoutintending to be bound by a specific mechanism, it is believed that onemethod for reducing the interaction of CD16A and immunoglobulinaggregates is by use of anti-CD16A antibodies, or other CD16A bindingproteins, to block the interaction.

Monoclonal antibody 3G8 (“mAb 3G8”) is a mouse monoclonal antibody thatbinds the Fc-binding domain of human CD16A and B with a K_(a) of1.times.10⁹ M⁻¹ (Fleit et al., 1982, Proc. Natl. Acad. Sci. U.S.A79:3275-79). 3G8 blocks the binding of human IgG₁ immune complexes toisolated human NK cells, monocytes and neutrophils, as well as toCD16A-transfected 293 cells. Experiments in which mAb 3G8 has beenadministered to human patients for treatment of idiopathicthrombocytopenic purpura (ITP) have been conducted (Clarkson et al.,1986, N. Engl. J. Med. 314:1236-39; Soubrane, et al., 1993, Blood81:15-19). Administration of the 3G8 antibody was reported to result inincreased platelet levels and was accompanied by one or more significantside effects, including a HAMA response, cytokine release syndrome,and/or pronounced neutropenia.

The present invention provides novel CD16A binding proteins, includinghumanized and/or aglycosylated monoclonal antibodies, and methods forreducing an deleterious immune response in a subject by administeringthe proteins. Administration of these binding proteins is shown to beprotective in well established models for two distinct autoimmunediseases: autoimmune hemolytic anemia (AHA) and idiopathicthrombocytopenic purpura. These results are indicative of efficacy ofthis treatment for other autoimmune diseases as well. Moreover, theinventors have discovered that, unexpectedly, administration ofanti-CD16A antibodies with altered effector function (e.g.,aglycosylated antibodies) protects against the deleterious immuneresponses characteristic of autoimmune disorders without inducing acutesevere neutropenia. Thus, the invention provides new reagents andmethods for antibody-mediated effected treatment of autoimmuneconditions without pronounced side-effects observed using alternativetreatments.

3. CD16A Binding Proteins

A variety of CD16A binding proteins may be used in the methods of theinvention. Suitable CD16A binding proteins include human or humanizedmonoclonal antibodies as well as CD16A binding antibody fragments (e.g.,scFv or single chain antibodies, Fab fragments, minibodies) and anotherantibody-like proteins that bind to CD16A via an interaction with alight chain variable region domain, a heavy chain variable regiondomain, or both.

In some embodiments, the CD16A binding protein for use according to theinvention comprises a V_(L) and/or V_(H) domain that has one or moreCDRs with sequences derived from a non-human anti-CD16A antibody, suchas a mouse anti-CD16A antibody, and one or more framework regions withderived from framework sequences of one or more human immunoglobulins. Anumber of non-human anti-CD16A monoclonal antibodies, from which CDR andother sequences may be obtained, are known (see, e.g., Tamm and Schmidt,1996, J. Imm. 157:1576-81; Fleit et al., 1989, p. 159; LEUKOCYTE TYPINGII: HUMAN MYELOID AND HEMATOPOIETIC CELLS, Reinherz et al., eds. NewYork: Springer-Verlag; 1986; LEUCOCYTE TYPING III: WHITE CELLDIFFERENTIATION ANTIGENS McMichael A J, ed., Oxford: Oxford UniversityPress, 1986); LEUKOCYTE TYPING IV: WHITE CELL DIFFERENTIATION ANTIGENS,Kapp et al., eds. Oxford Univ. Press, Oxford; LEUKOCYTE TYPING V: WHITECELL DIFFERENTIATION ANTIGENS, Schlossman et al., eds. Oxford Univ.Press, Oxford; LEUKOCYTE TYPING VI: WHITE CELL DIFFERENTIATION ANTIGENS,Kishimoto, ed. Taylor & Francis. In addition, as shown in the Examples,new CD16A binding proteins that recognize human CD16A expressed on cellscan be obtained using well known methods for production and selection ofmonoclonal antibodies or related binding proteins (e.g., hybridomatechnology, phage display, and the like). See, for example, O'Connel etal., 2002, J. Mol. Biol. 321:49-56; Hoogenboom and Chames, 2000, Imm.Today 21:371078; Krebs et al., 2001, J. Imm. Methods 254:67-84; andother references cited herein. Monoclonal antibodies from a non-humanspecies can be chimerized or humanized using techniques using techniquesof antibody humanization known in the art.

Alternatively, fully human antibodies against CD16A can be producedusing transgenic animals having elements of a human immune system (see,e.g., U.S. Pat. Nos. 5,569,825 and 5,545,806), using human peripheralblood cells (Casali et al., 1986, Science 234:476), by screening a DNAlibrary from human B cells according to the general protocol outlined byHuse et al., 1989, Science 246:1275, and by other methods.

It is contemplated that, for some purposes, it may be advantageous touse CD16A binding proteins that bind the CD16A receptor at the sameepitope bound by 3G8, or at least sufficiently close to this epitope toblock binding by 3G8. Methods for epitope mapping and competitivebinding experiments to identify binding proteins with the desiredbinding properties are well known to those skilled in the art ofexperimental immunology. See, for example, Harlow and Lane, cited supra;Stahl et al., 1983, Methods in Enzymology 9:242-53; Kirkland et al.,1986, J. Immunol. 137:3614-19; Morel et al., 1988, Molec. Immunol.25:7-15; Cheung et al., 1990, Virology 176:546-52; and Moldenhauer etal., 1990, Scand. J. Immunol. 32:77-82. Also see Examples and.sctn.3G(i), infra. For instance, it is possible to determine if twoantibodies bind to the same site by using one of the antibodies tocapture the antigen on an ELISA plate and then measuring the ability ofthe second antibody to bind to the captured antigen. Epitope comparisoncan also be achieved by labeling a first antibody, directly orindirectly, with an enzyme, radionuclide or fluorophore, and measuringthe ability of an unlabeled second antibody to inhibit the binding ofthe first antibody to the antigen on cells, in solution, or on a solidphase.

It is also possible to measure the ability of antibodies to block thebinding of the CD16A receptor to immune complexes formed on ELISAplates. Such immune complexes are formed by first coating the plate withan antigen such as fluorescein, then applying a specificanti-fluorescein antibody to the plate. This immune complex then servesas the ligand for soluble Fc receptors such as sFcRIIIa. Alternatively asoluble immune complex may be formed and labeled, directly orindirectly, with an enzyme radionuclide or fluorophore. The ability ofantibodies to inhibit the binding of these labeled immune complexes toFc receptors on cells, in solution or on a solid phase can then bemeasured.

CD16A binding proteins of the invention may or may not comprise a humanimmunoglobulin Fc region. Fc regions are not present, for example, inscFv binding proteins. Fc regions are present, for example, in human orhumanized tetrameric monoclonal IgG antibodies. As described in detailbelow, in some embodiments of the present invention, the CD16A bindingprotein includes an Fc region that has an altered effector function,e.g., reduced affinity for an effector ligand such as an Fc receptor orC1 component of complement compared to the unaltered Fc region (e.g., Fcof naturally occurring IgG₁, proteins). In one embodiment the Fc regionis not glycosylated at the Fc region amino acid corresponding toposition 297. Such antibodies lack Fc effector function.

Thus, in some embodiments of the invention, the CD16A binding proteindoes not exhibit Fc-mediated binding to an effector ligand such as an Fcreceptor or the C1 component of complement due to the absence of the Fcdomain in the binding protein while, in other cases, the lack of bindingor effector function is due to an alteration in the constant region ofthe antibody.

4. CD16A Binding Proteins Comprising CDR Sequences Similar to A mAb 3G8CDR Sequences

CD16A binding proteins that can be used in the practice of the inventioninclude proteins comprising a CDR sequence derived from (i.e., having asequence the same as or similar to) the CDRs of the mouse monoclonalantibody 3G8. Complementary cDNAs encoding the heavy chain and lightchain variable regions of the mouse 3G8 monoclonal antibody, includingthe CDR encoding sequences, were cloned and sequenced as described inthe Examples. The nucleic acid and protein sequences of 3G8 are providedbelow and are designated SEQ ID NO:1 and 2 (V_(H)) and SEQ ID NO:3 and 4(V_(L)). Using the mouse variable region and CDR sequences, a largenumber of chimeric and humanized monoclonal antibodies, comprisingcomplementary determining regions derived from 3G8 CDRs were producedand their properties analyzed. To identify humanized antibodies thatbind CD16A with high affinity and have other desirable properties,antibody heavy chains comprising a V_(H) region with CDRs derived from3G8 were produced and combined (by coexpression) with antibody lightchains comprising a V_(L) region with CDRs derived from 3G8 to produce atetrameric antibody for analysis. Properties of the resulting tetramericantibodies were determined as described below. As described below, CD16Abinding proteins comprising 3G8CDRs, such as the humanized antibodyproteins described hereinbelow, may be used according to the inventionto reduce a deleterious immune response.

A. V_(H) Region

In one aspect, the CD16A binding protein of the invention may comprise aheavy chain variable domain in which at least one CDR (and usually threeCDRS) have the sequence of a CDR (and more typically all three CDRS) ofthe mouse monoclonal antibody 3G8 heavy chain and for which theremaining portions of the binding protein are substantially human(derived from and substantially similar to, the heavy chain variableregion of a human antibody or antibodies).

In an aspect, the invention provides a humanized 3G8 antibody orantibody fragment containing CDRs derived from the 3G8 antibody in asubstantially human framework, but in which at least one of the CDRs ofthe heavy chain variable domain differs in sequence from thecorresponding mouse antibody 3G8 heavy chain CDR. For example, in oneembodiment, the CDR(S) differs from the 3G8 CDR sequence at least byhaving one or more CDR substitutions shown in Table 1 (e.g., valine atposition 34 in CDR1, leucine at position 50 in CDR2, phenylalanine atposition 52 in CDR2, tyrosine at position 52 in CDR2, aspartic acid atposition 52 in CDR2, asparagine at position 54 in CDR2, serine atposition 60 in CDR2, serine at position 62 in CDR2, tyrosine at position99 in CDR3, and/or aspartic acid at position 101 of CDR3). Suitable CD16binding proteins may comprise 0, 1, 2, 3, or 4, or more of thesesubstitutions (and often have from 1 to 4 of these substitutions) andoptionally can have additional substitutions as well.

In one embodiment, a CD16A binding protein may comprise a heavy chainvariable domain sequence that is the same as, or similar to, the V_(H)domain of the Hu3G8VH-1 construct, the sequence of which is provided inTable 4. For example, the invention provides a CD16A binding proteincomprising a V_(H) domain with a sequence that (1) differs from theV_(H) domain of Hu3G8VH-1 by zero, one, or more than one of the CDRsubstitutions set forth in Table 1; (2) differs from the V_(H) domain ofHu3G8VH-1 by zero, one or more than one of the framework substitutionsset forth in Table 1; and (3) is at least about 80% identical, often atleast about 90%, and sometimes at least about 95% identical, or even atleast about 98% identical to the Hu3G8VH-1 V_(H) sequence at theremaining positions.

Exemplary V_(H) domains of CD16 binding proteins of the invention havethe sequence of Hu3G8VH-5 and Hu3G8VH-22, as shown in Tables 4 and 6.

The V_(H) domain may have a sequence that differs from that of Hu3G8VH-1(Table 4) by at least one, at least two, at least three, at least four4, at least five, or at least six of the substitutions shown in Table 1.These substitutions are believed to result in increased affinity forCD16A and/or reduce the immunogenicity of a CD16A binding protein whenadministered to humans. In certain embodiments, the degree of sequenceidentity with the Hu3G8VH-1 V_(H) domain at the remaining positions isat least about 80%, at least about 90%, at least about 95% or at leastabout 98%.

TABLE 1 V_(H) Domain Substitutions Kabat No. Position RegionSubstitutions 1 2 FR1 Ile 2 5 FR1 Lys 3 10 FR1 Thr 4 30 FR1 Arg 5 34CDR1 Val 6 50 CDR2 Leu 7 52 CDR2 Phe or Tyr or Asp 8 54 CDR2 Asn 9 60CDR2 Ser 10 62 CDR2 Ser 11 70 FR3 Thr 12 94 FR3 Gln or Lys or Ala or His13 99 CDR3 Tyr 14 101 CDR3 Asp

For illustration and not limitation, the sequences of a number of CD16Abuilding protein V_(H) domains is shown in Table 4. As described in theExamples, infra, heavy chains comprising these sequences fused to ahuman Cγ1 constant region were coexpressed with the hu3G8VL-1 lightchain (described below) to form tetrameric antibodies, and binding ofthe antibodies to CD16A was measured to assess the effect of amino acidsubstitutions compared to the hu3G8VH-1 V_(H) domain. Constructs inwhich the V_(H) domain has a sequence of hu3G8VH-1, 2, 3, 4, 5, 8, 12,14, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 42, 43, 44 and 45 showed high affinity binding, withhu3G8VH-6 and -40 VH domains showing intermediate binding. CD16A bindingproteins comprising the VH domains of hu3G8VH-5 and hu3G8VH-22 areconsidered to have particularly favorable binding properties.

B. V_(L) Region

Similar studies were conducted to identify light chain variable domainsequences with favorable binding properties. In one aspect, theinvention provides a CD16A binding protein containing a light chainvariable domain in which at least one CDR (and usually three CDRs) hasthe sequence of a CDR (and more typically all three CDRs) of the mousemonoclonal antibody 3G8 light chain and for which the remaining portionsof the binding protein are substantially human (derived from andsubstantially similar to, the heavy chain variable region of a humanantibody or antibodies).

In one aspect, the invention provides a humanized 3G8 antibody orantibody fragment containing CDRs derived from the 3G8 antibody in asubstantially human framework, but in which at least one of the CDRs ofthe light chain variable domain differs in sequence from the mousemonoclonal antibody 3G8 light chain CDR. In one embodiment, the CDR(S)differs from the 3G8 sequence at least by having one or more amino acidsubstitutions in a CDR, such as, one or more substitutions shown inTable 2 (e.g., arginine at position 24 in CDR1; serine at position 25 inCDR1; tyrosine at position 32 in CDR1; leucine at position 33 in CDR1;aspartic acid, tryptophan or serine at position 50 in CDR2; serine atposition 53 in CDR2; alanine or glutamine at position 55 in CDR2;threonine at position 56 in CDR2; serine at position 93 in CDR3; and/orthreonine at position 94 in CDR3). In various embodiments, the variabledomain can have 0, 1, 2, 3, 4, 5, or more of these substitutions (andoften have from 1 to 4 of these substitutions) and optionally, can haveadditional substitutions as well.

In one embodiment, a suitable CD16A binding protein may comprise a lightchain variable domain sequence that is the same as, or similar to, theV_(L) domain of the Hu3G8VL-1 construct, the sequence of which isprovided in Table 5. For example, the invention provides a CD16A bindingprotein comprising a V_(L) domain with a sequence that (1) differs fromthe V_(L) domain of Hu3G8VL-1 by zero, one, or more of the CDRsubstitutions set forth in Table 2; (2) differs from the V_(L) domain ofHu3G8VL-1 by zero, one or more of the framework substitutions set forthin Table 2; and (3) is at least about 80% identical, often at leastabout 90%, and sometimes at least about 95% identical, or even at leastabout 98% identical to the Hu3G8VL-1 V_(L) sequence at the remainingpositions.

Exemplary V_(L) domains of CD16 binding proteins of the invention havethe sequence of Hu3G8VL-1 or Hu3G8VL-43, as shown in Tables 5 and 6.

The V_(L) domain may have a sequence that differs from that of Hu3G8VL-1(Table 5) by zero, one, at least two, at least 3, at least 4, at least5, at least 6, at least 7, at least 8, or at least 9 of thesubstitutions shown in Table 2. These substitutions are believed toresult in increased affinity for CD16A and/or reduce the immunogenicityof a CD16A binding protein when administered to humans. In certainembodiments, the degree of sequence identity at the remaining positionsis at least about 80%, at least about 90% at least about 95% or at leastabout 98%.

TABLE 2 V_(L) Domain Substitutions Kabat No. Position RegionSubstitutions 1 24 CDR1 Arg 2 25 CDR1 Ser 3 32 CDR1 Tyr 4 33 CDR1 Leu 550 CDR2 Asp or Trp or Ser 6 51 CDR2 Ala 7 53 CDR2 Ser 8 55 CDR2 Ala orGln 9 56 CDR2 Thr 10 93 CDR3 Ser 11 94 CDR3 Thr

For illustration and not limitation, the sequences of a number of CD16Abinding proteins V_(L) domains is shown in Table 5. As described in theExamples, infra, light chains comprising these sequences fused to ahuman Cκ constant domain were coexpressed with the Hu3G8VH-1 heavy chain(described above) to form tetrameric antibodies, and the binding of theantibodies to CD16A was measured to assess the effect of amino acidsubstitutions compared to the Hu3G8VL-1 V_(L) domain. Constructs inwhich the V_(L) domain has a sequence of hu3G8VL-1, 2, 3, 4, 5, 10, 16,18, 19, 21, 22, 24, 27, 28, 32, 33, 34, 35, 36, 37, and 42 showed highaffinity binding and hu3G8VL-15, 17, 20, 23, 25, 26, 29, 30, 31, 38, 39,40 and 41 showed intermediate binding. CD16A binding proteins comprisingthe V_(L) domains of hu3G8VL-1, hu3G8VL-22, and hu3G8VL-43 areconsidered to have particularly favorable binding properties.

C. Combinations of V_(L) and/or V_(H) Domains

As is known in the art and described elsewhere herein, immunoglobulinlight and heavy chains can be recombinantly expressed under conditionsin which they associate to produce a tetrameric antibody, or can be socombined in vitro. Similarly, combinations of V_(L) and/or V_(H) domainscan be expressed in the form of single chain antibodies, and still otherCD16A binding proteins that comprise a V_(L) and/or V_(H) domain can beexpressed by known methods. It will thus be appreciated that a3G8-derived V_(L)-domain described herein can be combined with a3G8-derived V_(H)-domain described herein to produce a CD16A bindingprotein, and all such combinations are contemplated.

For illustration and not for limitation, examples of useful CD16Abinding proteins are those comprising at least one V_(H) domain and atleast one V_(L) domain, where the V_(H) domain is from hu3G8VH-1,hu3G8VH-22 or hu3G8VH-5 and the V_(L) domain is from hu3G8VL-1,hu3G8VL-22 or hu3G8VL-43. In particular, humanized antibodies thatcomprise hu3G8VH-22 and either, hu3G8VL-1, hu3G8VL-22 or hu3G8VL-43, orhu3G8VH-5 and hu3G8VL-1 have favorable properties.

It will be appreciated by those of skill that the sequences of V_(LL)and V_(H) domains described here can be further modified by art-knownmethods such as affinity maturation (see Schier et al., 1996, J. Mol.Biol. 263:551-67; Daugherty et al., 1998, Protein Eng. 11:825-32; Boderet al., 1997, Nat. Biotechnol. 15:553-57; Boder et al., 2000, Proc.Natl. Acad. Sci. U.S.A. 97:10701-705; Hudson and Souriau, 2003, NatureMedicine 9:129-39). For example, the CD16A binding proteins can bemodified using affinity maturation techniques to identify proteins withincreased affinity for CD16A and/or decreased affinity for CD16B.

D. Constant Domains and Fc Region

As noted above, the CD16A binding protein of the invention may containlight chain and/or heavy chain constant regions (including the hingeregion connecting the C_(H)1 and C_(H)2 domains in IgG molecules). It iscontemplated that a constant domain from any type (e.g., IgM, IgG, IgD,IgA and IgE) of immunoglobulin may be used. The constant domain for thelight chain can be lambda or kappa. The constant domain for the heavychain can be any isotype (e.g., IgG₁, IgG₂, IgG₃ and IgG₄). Chimericconstant domains, portions of constant domains, and variants ofnaturally occurring human antibody constant domains (containingdeletions, insertions or substitutions of amino acid residues) may beused. For instance, a change in the amino acid sequence of a constantdomain can be modified to provide additional or different properties,such as altered immunogenicity or half-life of the resultantpolypeptide. The changes range from insertion, deletion or substitutionof a small number (e.g., less than ten, e.g., one, two, three or more)amino acid residues to substantial modifications of the constant regiondomain. Changes contemplated include those that affect the interactionwith membrane receptors, complement fixation, persistence incirculation, and other effector functions. For example, the hinge orother regions can be modified as described in U.S. Pat. No. 6,277,375.In particular, it will often be advantageous to delete or alter aminoacids of the Fc region. For example, the Fc region can be modified toreduce or eliminate binding to Fc effector ligands such as FcγRIII andthe C1q component of complement, such that the antibodies lack (or havesubstantially reduced) effector function. Antibodies having suchmodified Fc regions induce little or no antibody dependent cellularcytotoxicity (ADCC) and/or complement mediated lysis when administeredto a mammal, compared to unmodified antibodies. Assays to identifyantibodies lacking effector function are known in the art. See, e.g.,U.S. Pat. Nos. 6,194,551; 6,528,624; and 5,624,821, European Pat. No. EP0 753 065 B1, and PCT publication WO 00/42072.

The CD16A binding protein of the invention may include a human IgG₁ Fcdomain comprising one or more amino acid substitutions or deletions(relative to the parental naturally occurring IgG₁) that result in areduced interaction between the Fc domain of the binding protein andFcγRIIA and/or FcγRIIIA (e.g., to minimize potential activation ofmacrophages and/or minimize neutrophil diminution) and/or increasedbinding of the Fc region to FcγRIIB (e.g., to increase FcγRIIB-mediatedinhibition of effector cell activation; see Bolland and Ravetch, 1999,Adv. in Immunol. 72:149). Specific mutations effecting the desiredchanges in binding can be identified by selection using display ofmutant Fc libraries expressed on the surface of microorganisms, virusesor mammalian cells, and screening such libraries for mutant Fc variantshaving the desired property or properties. In addition, the literaturereports that particular residues or regions of the Fc are involved inFcγ interactions such that deletion or mutation of these residues wouldbe expected to result in reduced FcR binding. The binding site on humanantibodies for FcγR was reported to be the residues 233-239 (Canfield etal, 1991, J Exp Med 173:1483-91; Woof et al, 1986, Mol Imm. 23:319-30;Duncan et al., 1988, Nature 332:563). The crystal structure of FcγRIIIcomplexed with human IgG1 Fc revealed potential contacts between thereceptor and its ligand and also revealed that a single FcγRIII monomerbinds to both subunits of the Fc homodimer in an asymmetric fashion.Alanine-scanning mutagenesis of the Fc region confirmed the importanceof most of the predicted contact residues (Shields et al., 2001, J Biol.Chem. 276:6591-6604).

Exemplary Fc region mutations include, for example, L235E, L234A, L235A,and D265A, which have been shown to have low affinity for all FcR, intoCγ-1 (Clynes et al., 2000, Nat. Med. 6:443-46; Alegre et al., 1992, JImmunol 148:3461-68; Xu et al., 2000, Cell Immunol 200:16-26).Additional Fc region modifications purported to affect FcR binding aredescribed in WO 00/42072 (e.g., “class 4” Fc region variants) and WO02/061090.

Fc binding to FcγRIIA and FcγIIIA or other proteins can be measured byany of a number of methods, including ELISA to measure binding toisolated recombinant FcγR and RIA or FACS to measure binding to cells.Immune complexes and heat aggregated or chemically crosslinked Fc or IgGcan be used to test affinity for FcRs in such assays. In one embodiment,immune complexes are produced by expressing an Fc in the context of anFab with affinity for an antigen (such as fluorescein) and mixing theantibody and antigen to form an immune complex.

E. Fc Regions with Reduced Binding to Fc Effector Ligands Due toAglycosylation or Changes in Glycosylation

As discussed above, in CD16A binding proteins of the invention thatcomprise Fc domains (e.g., anti-CD16A monoclonal antibodies) the Fcdomain can be modified to achieve desired properties. In a particularaspect, the invention provides a CD16A binding protein, such as a humanor humanized anti-CD16A monoclonal antibody, comprising an Fc regionthat is not glycosylated. As demonstrated in Example 10, infra, theinventors have discovered that, unexpectedly, administration ofanti-CD16A antibodies with altered effector function (aglycosylatedantibodies) protects against autoimmune disorders without inducing acutesevere neutropenia. On the basis of this discovery, therapeuticanti-CD16A antibodies can be designed to protect against autoimmunediseases without inducing dangerous side effects.

In one embodiment, the invention provides a CD16A binding proteincomprising an Fc region derived from human IgG₁, where the amino acidscorresponding to position 297 of the C_(H)2 domains of the Fc region areaglycosyl. The terms “aglycosyl” or “aglycosylated,” when referring toan Fc region in its entirety, or a specific amino acid residue in the Fcregion, mean that no carbohydrate residues are attached to the specifiedregion or residue.

Human IgG antibodies that are aglycosylated show decreased binding to Fceffector ligands such as Fc receptors and C1q (see, e.g., Jefferis etal., 1995, Immunology Letters 44:111-17; Tao, 1989, J. of Immunology,143:2595-2601; Friend et al., 1999, Transplantation 68:1632-37; Radaevand Sun, 2001, J. of Biological Chemistry 276:16478-83; Shields et al,2001, J. of Biological Chemistry 276:6591-6604, and U.S. Pat. No.5,624,821). Without intending to be bound by a particular mechanism, itis believed that the aglycosylation of the amino acid at position 297 ofthe Fc domains of CD16A binding proteins described herein results inreduced binding to CD16A and the C1q component of complement. Suchaglycosylated antibodies lack effector function.

In human IgG₁ constant regions, the residue at position 297 isasparagine. In one embodiment of the present invention, the residue at,or corresponding to, position 297 of the Fc region of the CD16A bindingprotein is other than asparagine. Substitution of another amino acidresidue in the place of asparagine eliminates the N-glycosylation siteat position 297. Substitution of any amino acid residues which will notresult in glycosylation upon expression of the CD16A binding protein ina mammalian cell is appropriate for this embodiment. For instance, insome embodiments of the invention, the amino acid residue at position297 is glutamine or alanine. In some embodiments, the amino acid residueat position 297 is cysteine, which is optionally linked to PEG.

In other embodiments of the invention, the residue at position 297 mayor may not be asparagine, but is not glycosylated. This can beaccomplished in a variety of ways. For example, amino acid residuesother than the asparagine at position 297 are known to be important forN-linked glycosylation at position 297 (see Jefferis and Lund, 1997,Chem. Immunol. 65:111-28), and the substitution of residues at positionsother than position 297 of the C_(H)2 domain can result in a CD16Abinding protein aglycosylated at residue 297. For illustration and notlimitation, a residue at position 299 in the C_(H)2 domain that is otherthan threonine or serine will result in an antibody that isaglycosylated at position 297. Similarly, substitution of the amino acidat position 298 with proline will produce an antibody with anaglycosylated amino acid at position 297. In other embodiments of theinvention, Fc domains of IgG₂ or IgG₄ are used rather than IgG₁ domains.

Modification of the amino acid residues of CD16A binding proteins iswell within the ability of the ordinarily skilled practitioner, and canbe achieved by mutation of a polynucleotide encoding the binding proteinor portion thereof. The CD16A binding protein comprising an IgG-derivedFc region need not necessarily be mutated at the amino acid level to beaglycosylated. Binding proteins aglycosylated at position 297 of theIgG-derived Fc region can be produced by expressing the CD16A bindingprotein in certain cells (e.g., E. coli; see PCT publication WO02061090A2), cell lines or under certain cell culture growth conditionswhere glycosylation at Asn 297 does not take place. Alternatively,carbohydrate groups may be removed from a CD16A binding proteinfollowing expression of the protein, e.g., enzymatically. Methods forremoving or modifying carbohydrate groups on proteins are known andinclude use of endoglycosidases and peptide:N-glycosidases.

It will be apparent that a variety of methods can be used to modify theFc region of a CD16A binding protein to change its properties.Accordingly, unless otherwise specified, as used herein the term“modifying” in the context of modifying the Fc region of a CD16A bindingprotein includes modifying the protein itself directly, modifying thepolynucleotide that encodes the protein and/or modifying or selecting asuitable expression system production of the protein.

In addition to CD16A binding proteins that are aglycosylated at theposition corresponding to arginine 297, variants with reduced binding toFc effector ligands due to only partial removal, or modification, of thecarbohydrate at that position may be used in the present invention. Forexample, the Fc region can be modified to include a non-naturallyoccurring carbohydrate that does not bestow binding protein witheffector function. As used herein, a “modified Fc region” is an Fcregion that has been derived from a parent Fc region, but which differsin glycosylation pattern from the parent Fc region.

F. Production of CD16A Binding Proteins

CD16A binding proteins of the invention can be produced using a varietyof methods well known in the art, including de novo protein synthesisand recombinant expression of nucleic acids encoding the bindingproteins. The desired nucleic acid sequences can be produced byrecombinant methods (e.g., PCR mutagenesis of an earlier preparedvariant of the desired polynucleotide) or by solid-phase DNA synthesis.Usually recombinant expression methods are used. In one aspect, theinvention provides a polynucleotide that comprises a sequence encoding aCD16A binding protein disclosed herein or a CD16A binding fragmentthereof, for example a sequence encoding a V_(L) or V_(H) describedherein, or antibody heavy chain or light chain described herein. Becauseof the degeneracy of the genetic code, a variety of nucleic acidsequences encode each immunoglobulin amino acid sequence, and thepresent invention includes all nucleic acids encoding the bindingproteins described herein.

Recombinant expression of antibodies is well known in the art and can becarried out, for example, by inserting nucleic acids encoding light andheavy chain variable regions, optionally linked to constant regions,into expression vectors. Expression vectors typically include controlsequences such as a promoter, an enhancer, and a transcriptiontermination sequence to which DNA segments encoding polypeptides (e.g.,immunoglobulin chains) are operably linked to ensure the expression ofimmunoglobulin polypeptides. Expression vectors are typically replicablein the host organisms either as episomes or as an integral part of thehost chromosomal DNA. The light and heavy chains can be cloned in thesame or different expression vectors.

Immunoglobulin light and heavy chains are expressed using standardmethods. A multiple polypeptide chain antibody or antibody fragmentspecies can be made in a single host cell expression system wherein thehost cell produces each chain of the antibody or antibody fragment andassembles the polypeptide chains into a multimeric structure to form theantibody or antibody fragment in vivo. See e.g., Lucas et al., 1996,Nucleic Acids Res., 24:1774-79. When heavy and light chains are clonedon separate expression vectors, the vectors are co-transfected to obtainexpression and assembly of intact immunoglobulins. Alternatively,recombinant production of antibody heavy and light chains in separateexpression hosts followed by assembly of antibody from separate heavyand light chains in vitro is known. See, e.g., U.S. Pat. No. 4,816,567and Carter et al., 1992, Bio/Technology 10:163-67.

The CD16A binding proteins are conveniently expressed in procaryotic oreukaryotic cells. Useful hosts for antibody expression include bacteria(see, e.g., PCT publication WO 02/061090), yeast (e.g., Saccharomyces),insect cell culture (Putlitz et al., 1990, Bio/Technology 8:651-54),plants and plant cell cultures (Larrick and Fry, 1991, Hum. AntibodiesHybridomas 2:172-89), and mammalian cells. Methods for expression arewell known in the art. For example, in E. coli, vectors using the lacpromoter to drive expression of heavy fd and light chains fused tovarious prokaryotic secretion signal sequences such as pe1B haveresulted in successful secretion of scFv and Fab fragments into theperiplasmic space or into the culture medium (Barbas et al., 1991, Proc.Natl. Acad. Sci. U.S.A 88:7978-82). A vector derived from pET25b inwhich the lac promoter has been inserted in place of the T7 promoter maybe used.

Mammalian cells are especially useful for producing CD16A bindingproteins, including tetrameric antibodies or fragments thereof. A numberof suitable host cell lines capable of secreting intact heterologousproteins are known, and include CHO cell lines, COS cell lines, HeLacells, L cells and myeloma cell lines. Expression vectors for mammaliancells can include expression control sequences, such as an origin ofreplication, a promoter, an enhancer, ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Examples of expression control sequences are promoters derived fromendogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. In one embodiment, binding proteins areexpressed using the CMV immediate early enhancer/promoter in the vectorpcDNA3.1 or a similar vector. To facilitate secretion, the genes can befused to a gene cassette containing the signal sequence of a mouse V_(H)gene described by Orlandi et al., 1989, Proc. Natl. Acad. Sci. U.S.A86:3833-37, which has been widely used for high-level secretion ofimmunoglobulins.

The vectors containing the DNA segments encoding the polypeptides ofinterest can be transferred into the host cell using routine, dependingon the type of cellular host. For example, calcium chloride transfectionis commonly utilized for prokaryotic cells, whereas calcium phosphatetreatment, electroporation, lipofection, biolistics or viral-basedtransfection may be used for other cellular hosts. Other methods used totransform mammalian cells include the use of polybrene, protoplastfusion, liposomes, electroporation, and microinjection (see generally,Sambrook et al., supra). For transient expression, cells, e.g., HEK293,can be co-transfected with separate heavy and light chain expressionvectors using a cationic lipid (e.g., Lipofectamine 2000, Invitrogen).This method can achieve expression levels of 10-20 mg/l of IgG inconditioned medium after 3 days. The cells can then be re-fed andsimilar quantities harvested after 3 more days. It will be appreciatedthat, for some uses, the cells expressing CD16A binding proteins can bemaintained in medium containing FBS screened for very low levels ofbovine IgG, or, alternatively, in serum-free medium.

In addition to expression of tetrameric antibodies, single chainantibodies, antibody fragments, and other CD16A binding proteins can beprepared. For example, immunoglobulin fragments can be prepared byproteolytic digestion of tetrameric antibodies, or more often, byrecombinant expression of truncated antibody constructs. Usually, singlechain V region (“scFv”) constructs are made by linking V_(L) and/orV_(H) domain using a short linking peptide (see, e.g., Bird et al.,1988, Science 242:423-26; U.S. Pat. Nos. 4,946,778; 5,455,030;6,103,889; and 6,207,804).

Once expressed, the binding proteins can be purified using procedureswell known in the art, including ammonium sulfate precipitation,affinity chromatography, gel electrophoresis and the like (see,generally, Harris and Angal, 1990, PROTEIN PURIFICATION APPLICATIONS, APRACTICAL APPROACH Oxford University Press, Oxford, UK; and Coligan etal., supra). In one embodiment, purification is accomplished bycapturing the antibody using a high flow rate protein A resin such asPoros A (Perseptive Biosystems, Inc), and elution at low pH, followed bysize exclusion chromatography to remove any traces of aggregate present.Since FcγRIIIA binds preferentially to aggregated IgG, removal ofaggregates will be desirable for certain applications. The bindingproteins can be purified to substantial purity if desired, e.g., atleast about 80% pure, often at least about 90% pure, more often leastabout 95%, or at least about 98% pure. In this context, the percentpurity is calculated as a weight percent of the total protein content ofthe preparation, and does not include constituents which aredeliberately added to the composition after the binding protein ispurified.

CD16A binding proteins can be modified after expression. For example,derivation of antibodies with polyethylene glycol (“pegylation”) isreported to increase residence time (half-life and stability) and reduceimmunogenicity in vivo without alteration of biological activity. See,e.g., Leong et al., 2001, Cytokine 16:106-19; Koumenis et al., 2000, IntJ Pharm 198:83-95; U.S. Pat. No. 6,025,158. CD16A binding proteins canbe conjugated to a detectable label or ligand (e.g., a radioisotope orbiotin). Other modifications are well known in the art and are alsocontemplated.

G. Properties of CD16A Binding Proteins

In certain embodiments, CD16A binding proteins having properties asdescribed below are used in the methods of the invention.

i) Binding Affinity

CD16A binding proteins can be described by reference to their bindingproperties and biological activity. In various embodiments, the bindingconstant for the interaction of a CD16A binding protein of the inventionand CD16A is between 0.1 and 5 nM, less than about 2.5 nM, less thanabout 1 nM, or less than about 0.5 nM. Usually the binding protein bindsCD16A with an affinity that is within 4-fold, optionally within 2-fold,of the binding affinity exhibited under similar conditions by 3G8 or thechimeric antibody comprising the heavy chain Ch3G8V_(H) and the lightchain Ch3G8V_(L) as described herein below. In an embodiment, thebinding affinity for CD16A is greater than that of 3G8. In analternative embodiment, the binding affinity for CD16B is no greaterthan, and preferably less than, 3G8 or the chimeric antibody Ch3G8.

Binding can be measured using a variety of methods, including ELISA,biosensor (kinetic analysis), and radioimmunoassay (RIA). ELISA is wellknown (see, Harlow and Lane, supra, and Ausubel et al., supra) and canbe carried out using conditioned medium containing binding proteins or,alternatively, with purified antibodies. The concentration of antibodythat results in 50% apparent maximal binding provides an estimate ofantibody Kd.

Binding can also be detected using a biosensor assay, which alsoprovides information on the kinetic and equilibrium properties ofantibody binding to FcγRIIIA. An exemplary biosensor assay uses theBIAcore system (Malmqvist et al., 1997, Curr. Opin. Chem. Biol.1:378-83). The BIAcore system relies on passing analyte over a sensorchip onto which the ligand (e.g., CD16A) is immobilized. The binding ofthe analyte can be measured by following surface plasmon resonance (SPR)signal, which changes in direct proportion to the mass bound to thechip. A fixed concentration of analyte is passed over the chip for aspecific amount of time, allowing for the measurement of the associationrate, k(on). Following this phase, buffer alone is passed over the chipand the rate at which the analyte dissociates from the surface, k(off)can be measured. The equilibrium dissociation constant can be calculatedfrom the ratio of the kinetic constants; Kd=k(on)/k(off).

A radioimmunoassay (RIA) can be used to measure the affinity ofantibodies for FcγRIII-bearing cells, and to measure inhibition of IgGcomplexes to cells by these antibodies. In an exemplary assay, ¹²⁵Ilabeled binding protein is prepared and specific radioactivity of theprotein determined. Labeled binding protein and cells are mixed forseveral hours, the cells and bound material are separated from theunbound material by centrifugation, and the radioactivity in bothcompartments is determined. A direct binding format is used to determinethe Kd of, and the number of binding sites for, iodinated bindingprotein using Scatchard analysis of the binding data. Controlscontaining an excess of cold (unlabeled) binding protein competitor canbe included to ensure the results reflect specific interactions.Examples of suitable cells include (1) NK cells or macrophages derivedfrom normal human peripheral blood lymphocytes; (2) Cells obtained fromhuCD16A transgenic mice (Li, 1996 J. Exp. Med. 183:1259-63); (3)mammalian cell lines expressing the extracellular portion of CD16A fusedto the transmembrane and intracellular domain of RII or another receptor(such as CD8 or LFA-3); (4) mammalian cell lines (e.g., CHO, HEK-293,COS) transfected transiently or stably with CD16A expression vectors(and optionally coexpressing gamma chain for optimal expression receptorexpression).

Examples of expression vectors useful for expression of CD16A and otherpolypeptides for use in binding assays include mammalian expressionvectors (e.g., pcDNA 3.1 or pCI-neo) that contain a strongpromoter/enhancer sequence (e.g., CMV immediate early) and apolyadenylation/transcription termination site flanking a polylinkerregion into which the CD16A gene is introduced. Usually the vectorincludes a selectable marker such as a neomycin resistance gene.

In one embodiment, the CD16A expressed for use in assays has thesequence: MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNTTITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNTRSSTRDWKDHKFKWRKDPQDK (SEQ ID NO:116). CD16A with the sequence:MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK (SEQ ID NO:117) can also be used.Additional CD16A variants and substitutes will be known to, or readilydiscernible from the scientific literature by, the ordinarily skilledreader.

Competitive assay formats can be used to measure the ability of a CD16Abinding protein to inhibit binding of another molecule to the receptor.For example, in one competitive assay format a fixed amount of labeled3G8 is mixed with varying amounts of either unlabeled 3G8, CD16A bindingprotein or an irrelevant IgG (control) and added to FcγRIIIA expressingcells. After incubation and separation of the cell-bound material fromthe material free in solution, the amount of bound labeled 3G8 (and/oroptionally also the unbound labeled 3G8) is determined. Theconcentration of unlabeled mAb which results in a 50% decrease in thebinding of labeled 3G8 (IC50) is then determined from this data.

ii. Blocking Immune Complex Binding to FcγRIIIA

Another characteristic of the CD16A binding proteins of the invention isthe ability to inhibit binding of immune complexes to CD16A (“ICBlocking Activity”). Usually the binding protein has IC BlockingActivity that is within 4-fold, preferably within 2-fold, of theactivity exhibited under similar conditions by 3G8 or the chimericantibody, Ch3G8, described herein.

Assays for measuring ability of an antibody to block binding ofcomplexed IgG to CD16 are known. See, e.g., Knapp et al, 1989, LEUKOCYTETYPING IV, Oxford University Press, Oxford, p. 574-97; and Edberg andKimberly, 1997, J Immunol 159:3849-57. One suitable assay is an RIAassay with the format described above for the competitive assay, butsubstituting ¹²⁵I-labeled aggregated irrelevant human IgG₁ for the¹²⁵I-labeled 3G8 used in the competitive assay described above.

The invention provides a method of inhibiting the binding of IgGantibodies to CD16 on a cell by contacting the cell with a CD16A bindingprotein under conditions in which the CD16A binding protein binds theFcγRIII on the cell. The contacting can be in vivo (e.g., byadministering the binding protein in a mammal) or in vitro (e.g., byaddition of antibodies to cultured cells expressing the FcγRIII). IgGantibodies that are inhibited from binding the FcγRIII can beadministered to the animal or added to a culture medium before or afteraddition or administration of the binding protein, or may be present inan animal normally or in response to a disease state. In one embodiment,the CD16 on the surface of the cell is CD16A.

iii. In Vivo Protection Against Platelet Depletion

The ability of the CD16A binding proteins of the invention to reducedeleterious immune responses can be assessed in a variety of animalmodels. An exemplary model system is a mouse model for idiopathicthrombocytopenic purpura (ITP) (see, Oyaizu et al., 1988, J Exp.Med.167:2017-22; Mizutani et al, 1993, Blood 82:837-44). See Example 9,infra. Other suitable models are known in the art. Other animal modelsinclude rodent models of inflammatory diseases described in, forexample, Current Protocols in Immunology (in some cases modified byusing animals transgenic for human CD16A). Transgenic mice can be madeusing routine methods or can be purchased from commercial sources (e.g.,Taconic Inc., German Town N.Y.).

A example of a procedure suitable for assessing the ability of a CD16Abinding protein to provide protection from platelet depletion in a mousemodel is described in Example 8, infra. CD16A binding proteins can beadministered to muFcγRIII−/−, huFcγRIIIA transgenic mice at a variety ofconcentrations, and ITP subsequently induced in the mice (e.g., byadministering the 6A6 or chimeric 6A6 antibody) to the mice. At timedintervals after the administration of 6A6/ch6A6, the mice are bled andthe platelet counts are determined. Optionally, the IC₅₀ for eachmolecule is then determined at the time point where maximal plateletdepletion is observed in the negative control group. Based on theresults of Example 8 and on prior studies, maximum depletion occurred2-6 hr after 6A6 administration. IC₅₀s are determined graphically, usinga curve-fitting program such as the four-parameter fit provided in theSigmaPlot program. Statistically significant inhibition of depletion ofplatelets after administration of 6A6 in the treatment group compared tothe untreated group and a group administered an identical formulation ofan irrelevant, isotype matched mAb is indicative of the desiredbiological activity.

Experiments in which protection by CD16A binding proteins was assayedare described in the Examples, infra. Preparations of recombinant mouse3G8 produced in HEK-293 cells, chimeric 3G8 with human IgG1 or IgG2constant domains (ch3G8-γ1 produced in HEK-293 and CHO-K1 cells, andch3G8-γ2 produced in HEK-293 cells), and a ch3G8-γ1 variant (ch3G8-γ1D265A) did not provide significant protection. Murine 3G8, produced fromthe hybridoma, and a chimeric version of 3G8 containing an aglycosylatedhuman G1 constant region (Ch3G8-G1 N297Q), produced in HEK-293 cells,were able to protect animals from platelet depletion in the mouse model.As shown in Example 10, 11 and 15-17, infra, Ch3G8 N297Q andaglycosylated humanized antibodies protected against platelet depletionin the ITP mouse model. Although not intending to be bound by aparticular theory, one possibility is that since ch3G8 N297Q is largelydevoid of effector function, it is more efficient than ch3G8 inprotecting mice against ITP. Thus, these data suggest that anti-CD16Aantibodies without effector function (e.g., aglycosylated antibodies)have advantages compared to some glycosylated (e.g., glycosylatedrecombinant) antibodies. Further, as described in the examples,administration of aglycosylated anti-CD16A antibody to muFcgRIII−/−,huFcRIIIB transgenic mice did not result in neutrophil depletion in theblood, spleen, and bone marrow. Without intending to be bound by aparticular theory, there are several possible explanations for theseunexpected results. Protein glycosylation is known to vary in differentcell lines, especially those from different species. A difference in thenature of the carbohydrate attached to the antibody constant region as aconsequence of expression in different cell types may be responsible forthe difference in activity, i.e., if the lack of activity results inpart from effector cell activation caused by ch3G8 binding to Fcreceptors (or complement) via the antibody Fc region in aglycosylation-dependent manner. Alternatively, recombinant murine andch3G8 may contain other post-translational modifications that affectactivity and which can be eliminated by using different cell lines toexpress the CD16A binding proteins. It is possible that a combination ofisotype and/or isotype containing mutations to eliminate effectorfunction may provide similar protective effects as elimination of thecarbohydrate on the Fc.

5. Methods of Treatment

A number of diseases and conditions characterized by an deleteriousimmune response can be treated using the binding proteins of theinvention a CD16A binding protein as described herein (e.g., comprisinga V_(L) and/or V_(H) sequence as disclosed herein and, optionally, a Fcregion modified as disclosed herein to have a reduced effectorfunction). In one embodiment, the binding protein is administered to asubject with an autoimmune disease (i.e., a disease characterized by theproduction of autoantibodies). It is believed that pathogenic IgGantibodies observed in autoimmune diseases are either the pathogenictriggers for these diseases or contribute to disease progression andmediate disease through the inappropriate activation of cellular Fcreceptors. Aggregated autoantibodies and/or autoantibodies complexedwith self antigens (immune complexes) bind to activating FcRs, therebytriggering the pathogenic sequelae of numerous autoimmune diseases(which occur in part because of immunologically mediated inflammationagainst self tissues). Without intending to be bound by a particularmechanism of action, the CD16A binding proteins described hereininterfere with and reduce the interaction of the autoimmune antibodiesand FcγRIII receptors.

Examples of autoimmune diseases that can be treated include, withoutlimitation, idiopathic thrombocytopenic purpura (ITP), rheumatoidarthritis (RA), systemic lupus erythrematosus (SLE), autoimmunehemolytic anemia (AHA), scleroderma, autoantibody triggered urticaria,pemphigus, vasculitic syndromes, systemic vasculitis, Goodpasture'ssyndrome, multiple sclerosis (MS), psoriatic arthritis, ankylosingspondylitis, Sjogren's syndrome, Reiter's syndrome, Kowasaki's disease,polymyositis and dermatomyositis. Other examples of diseases orconditions that can be treated according to the invention also includeany diseases susceptible to treatment with intravenous immunoglobulin(IVIG) therapy (e.g., allergic asthma). Thus, the treatment ofautoimmune diseases heretofore treated by IVIG therapy (in oneembodiment, a condition other than ITP) is contemplated. While detailedunderstanding of the mechanism of action of IVIG has not beenestablished, it is proposed that modulating the activity of cellularFcγRs plays a role in its in vivo efficacy. The protective activity ofIVIG may rely on the small percentage of dimeric or polymeric IgGpresent in the preparation. The specificity of the FcγRIII pathway incoupling cytotoxic and immune complex antibodies to effector responsesand the ability to directly block this pathway with a mAb stronglysuggests that an anti-FcγRIII antibody will have enhanced activityrelative to IVIG.

A reduction in a deleterious immune response can be detected as areduction in inflammation. Alternatively, a reduction in a deleteriousimmune response can be detected as a reduction in symptomscharacteristic of the condition being treated (e.g., a reduction insymptoms exhibited by a subject suffering from an autoimmune condition),or by other criteria that will be easily recognized by physicians andexperimentalists in the field of automimmunity. It will be apparentthat, in many cases, specific indicia of reduction will depend on thespecific condition being treated. For example, for illustration and notlimitation, a reduction in a deleterious immune response in a subjectwith ITP can be detected as a rise in platelet levels in the subject.Similarly, a reduction in a deleterious immune response in a subjectwith anemia can be detected as a rise in RBC levels in the subject. Aclinician will recognize significant changes in platelet or RBC levels,or other reponses following treatment.

The deleterious immune response is optionally due to idiopathicthrombocytopenic purpura resulting from the administration of anantiplatelet antibody, optionally murine monoclonal antibody 6A6, to amuFcγRIII−/−, huFcγRIIIA transgenic mouse.

In one aspect, the invention provides a method for treating anautoimmune disease, such as ITP, by administering a CD16A bindingprotein that is largely devoid of effector function. In an embodiment,the CD16A binding protein comprises Fc regions derived from human IgG.In an embodiment, the Fc regions are aglycosyl. In an embodiment, theposition 297 of each of the C_(H)2 domains is a residue of thanasparagine or proline. In one aspect, the binding protein comprises avariable region sequence as described elsewhere herein. However, asdiscussed herein, the compositions and treatment methods of theinvention are not limited to specific CD16A binding proteins derivedfrom murine mAb 3G8, but are applicable to CD16A binding proteins ingeneral. In an embodiment, the CD16A binding protein is a tetramericantibody protein having two light chains and two heavy chains.

In a related aspect, the invention provides methods of reducing andeleterious immune response in a mammal without significantly reducingneutrophil levels or inducing neutropenia (e.g., severe neutropenia ormoderate neutropenia) by administering to the mammal a therapeuticallyeffective amount of a pharmaceutical composition comprising a CD16Abinding protein described herein. In an embodiment, the mammal is human.In an embodiment, the mammal is a nonhuman mammal (e.g., mouse)comprising one or more human transgenes.

For therapeutic applications, the binding proteins of the invention areformulated with a pharmaceutically acceptable excipient or carrier,e.g., an aqueous carrier such as water, buffered water, 0.4% saline,0.3% glycine and the like, optionally including other substances toincrease stability, shelf-life or to approximate physiologicalconditions (sodium acetate, sodium chloride, potassium chloride, calciumchloride, sodium lactate, histidine and arginine). For administration toan individual, the composition is preferably sterile, and free ofpyrogens and other contaminants. The concentration of binding proteincan vary widely, e.g., from less than about 0.01%, usually at leastabout 0.1% to as much as 5% by weight. Methods for preparing parentallyadministrable compositions are known or apparent to those skilled in theart and are described in more detail in, for example, Remington, THESCIENCE OF PRACTICE AND PHARMACY, 20th Edition Mack Publishing Company,Easton, Pa., 2001). The pharmaceutical compositions of the invention aretypically administered by a parenteral route, most typicallyintravenous, subcutaneous, intramuscular, but other routes ofadministration can be used (e.g., mucosal, epidermal, intraperitoneal,oral, intranasal, and intrapulmonary). Although not required,pharmaceutical compositions are preferably supplied in unit dosage formsuitable for administration of a precise amount. In one embodiment,CD16A binding proteins can be administered in a form, formulation orapparatus for sustained release (e.g., release over a period of severalweeks or months).

In one embodiment, polynucleotides encoding CD16A binding proteins(e.g., CD16A binding protein expression vectors) are administered to apatient. Following administration, the CD16A binding protein isexpressed in the patient. Vectors useful in administration of CD16Abinding proteins can be viral (e.g., derived from adenovirus) ornonviral. Usually the vector will comprise a promoter and, optionally,an enhancer that serve to drive transcription of a protein or proteins.Such therapeutic vectors can be introduced into cells or tissues invivo, in vitro or ex vivo. For ex vivo therapy, vectors may beintroduced into cells, e.g., stem cells, taken from the patient andclonally propagated for autologous transplant back into the same patient(see, e.g., U.S. Pat. Nos. 5,399,493 and 5,437,994).

The compositions can be administered for prophylactic and/or therapeutictreatments. In prophylactic applications, compositions are administeredto a patient prior to an expected or potential deleterious immuneresponse. For example, idiopathic thrombocytopenic purpura and systemiclupus erythrematosus are conditions in which an deleterious immuneresponse can be exacerbated by administration of certain medications.The CD16A binding compositions of the invention can be administered inanticipation of such medication-induced responses to reduce themagnitude of the response. In therapeutic applications, compositions areadministered to a patient already suffering from an deleterious immuneresponse in an amount sufficient to at least partially ameliorate thecondition and its complications. An amount adequate to accomplish thismay be a “therapeutically effective amount” or “therapeuticallyeffective dose.” Amounts effective for these uses depend upon theseverity of the condition and the general state of the patient's ownimmune system, but generally range from about 0.01 to about 100 mg ofantibody per dose, with dosages of from 0.1 to 50 mg and 1 to 10 mg perpatient being more commonly used. An “inflammation reducing amount” ofthe binding protein can also be administered to a mammal to reduce adeleterious immune response.

The administration of the CD16A binding proteins can be administeredaccording to the judgement of the treating physician, e.g., daily,weekly, biweekly or at any other suitable interval, depending upon suchfactors, for example, as the nature of the ailment, the condition of thepatient and half-life of the binding protein.

CD16A binding proteins can be administered in combination othertreatments directed to alleviation of the deleterious immune response orits symptoms or sequalae. Thus, the binding proteins can be administeredas part of a therapeutic regimen that includes co-administration ofanother agent or agents, e.g., a chemotherapeutic agent such as anon-steroidal anti-inflammatory drug (e.g., aspirin, ibuprofen),steroids (e.g., a corticosteroid, prednisone), immunosuppressants (e.g.,cyclosporin A, methotrexate cytoxan), and antibodies (e.g., inconjunction with IVIG).

6. Increasing the Therapeutic Efficacy of a CD16A Binding Protein

In a related aspect, the invention provides a method for increasing thetherapeutic efficacy of a CD16A binding protein comprising one or moreFc domains (e.g., anti-CD16A antibodies comprising two Fc domains) bymodifying the protein so that it has Fc region(s) with reduced bindingto at least one Fc effector ligand compared to the original (i.e.,unmodified) Fc region. For example, the Fc region can be modified sothat the Fc region is not glycosylated. As described above, modificationof the Fc region can be accomplished in several ways (e.g., by geneticmutation, by choice of expression system to change the Fc glycosylationpattern, and the like). In one embodiment, the Fc effector ligand isFcγRIII. In one embodiment, the Fc effector ligand is the C1q componentof complement. As used in this context, a subject CD16A binding proteinhas increased “therapeutic efficacy” compared to a reference bindingprotein that induces neutropenia when administered if the subject CD16Abinding protein does not induce neutropenia (or results in less severeneutropenia). For example, a CD16A binding protein that reduces theseverity of an deleterious immune response (e.g., ITP or experimentallyinduced ITP in a mammal) and reduces neutrophil levels in the animal byx % has greater “therapeutic efficacy” than a CD16A binding protein thatreduces the severity of an deleterious immune response and reducesneutrophil levels in the animal by y %, if y is greater than x, e.g.two-fold greater. In one embodiment, the protein is modified by mutationsuch that the modified protein is aglycosylated.

For example, the invention provides methods for producing a modifiedCD16 binding protein comprising a modified immunoglobulin heavy chain,the modified CD16 binding protein having greater therapeutic efficacythan a parent CD16 binding protein comprising a parent immunoglobulinheavy chain, by (i) introducing at least one mutation into a parentpolynucleotide that encodes the parent immunoglobulin heavy chain toproduce a modified polynucleotide that encodes the modifiedimmunoglobulin heavy chain, the mutation introducing into the modifiedimmunoglobulin heavy chain an amino acid substitution that changes,reduces or eliminates glycosylation in the C_(H)2 domain of the parentimmunoglobulin heavy chain; and (ii) expressing the modifiedpolynucleotide in a cell as the modified immunoglobulin heavy chain soas to produce the modified CD16 binding protein heavy chain. Optionally,the heavy chain is produced under conditions of co-expression with alight chain to produce a tetrameric antibody.

7. EXAMPLES Example 1 Mouse 3G8 VH and VL and Chimeric MoleculesGenerated Therefrom

A) Mouse 3G8 VH and VL

The cDNA encoding the mouse 3G8 antibody light chain was cloned. Thesequence of the 3G8 antibody heavy chain was provided by Dr. JeffryRavetch. The amino acid sequences of the 3G8 V_(H) and V_(L) areprovided in Tables 4 and 5, infra. Nucleic acid sequences encoding thevariable regions are:

SEQ ID NO: 1 {3G8VH} CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGGACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTAGAGTGGCTGGCACACATTTGGTCGGATGATGACAAGCGCTATAATCCAGCCCTCAAGAGCCGACTGACAATCTCCAACGATACCTCCAGCAACCAGGTATTCCTCAAAATCGCCAGTGTGGACACTGCAGATACTGCCACATACTACTGTGCTCAAATAAACCCCGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTC TGCA SEQ ID NO: 3{3G8VL} GACACTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTTTGATGGTGATAGTTTTATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATACTACATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGCCAGTGCGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATACTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA

B) Chimeric Heavy Chain

To create a chimeric gene coding for expression of the mouse 3G8 V_(H)fused to a human constant domain, the nucleic acid encoding the 3G8V_(H) was fused to sequences encoding a signal peptide (see Orlandi etal., 1989, Proc. Natl. Acad. Sci. U.S.A 86:3833-37; in lower caseunderline below) and a human Cγ1 constant region (in lower case below)using standard techniques (including overlapping PCR amplification). Tofacilitate cloning, a SacI site was introduced, resulting in a singleresidue change in VH FR4 (ala→ser). This change in FR4 does not affectbinding to CD16. The resulting nucleic acid had the sequence shownbelow. The regions encoding the V_(H) domain is in upper case.

SEQ ID NO: 5 {ch3G8VH}gctagcgtttaaacttaagcttgttgactagtgagatcacagttctctctacagttactgagcacacaggacctcaccatgggatggagctgtatcatcctcttcttggtagcaacagctacaggtaaggggctcacagtagcaggcttgaggtctggacatatatatgggtgacaatgacatccactttgcctttctctccacagqgtccactccCAGGTTACCCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTCACTTGTTCTTTCTCTGGGTTTTCACTGAGGACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTAGAGTGGCTGGCACACATTTGGTGGGATGATGACAAGCGCTATAATCCAGCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCAGCAACCAGGTATTCCTCAAAATCGCCACTGTGGACACTGCAGATACTCCCACATACTACTGTGCTCAAATAAACCCCGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTGAGCTCAgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtccgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagag cctctccctgtctccgggtaaatgagtgcggccgcgaattc

This construct was inserted into the pCI-Neo (Promega Biotech) at theNheI-EcoRI sites of the polylinker for use for expression of thechimeric heavy chain in cells.

C) Chimeric Light Chain

To create a chimeric gene coding for the mouse 3G8 V_(L) fused to ahuman constant domain, this 3G8 V_(L) segment was fused to a signalsequence (as for the V_(H) above; (lower case underlined) and a human Cκconstant region (lower case) cDNA using standard techniques, resultingin a nucleic acid with the sequence shown below:

SEQ ID NO: 6 ch3G8VL gctagctgagatcacagttctctctacagttactgagcacacaggacctcaccatgggatggagctgtatcatcctcttcttggtagcaacagctacaggtaaggggctcacgtagcaggcttgaggtctggacatatatatgggtgacaatgacatccactttgcctttctctccacaggtgtccactccGACACTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTTTGATGGTGATAGTTTTATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATACTACATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGCCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATACTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTTGAGATCAAAcgaactgtggctgcaccatcggtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcaatacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttagttctagagtcgactctagaggatccccgggtaccgagctcgaattc

This construct was inserted into pCI-Neo (Promega Biotech) at theNheI-EcoRI sites of the polylinker for use for expression of thechimeric light chain in cells.

D) Expression

The ch3G8VH and ch3G8VL chimeric proteins described above can beco-expressed to form a chimeric antibody, referred to as ch3G8. Thechimeric antibody ch3G8 can be expressed either in a myeloma or in othermammalian cells (e.g., CHO, HEK-293). An example of a procedure forexpression of CD116A binding proteins such as ch3 G8 and variants isprovided in Example 4, infra.

Example 2 Humanized Anti-CD16A Binding Proteins

A) Humanized Heavy Chain

CDR encoding sequences from the mouse 3G8 V_(H) clone were fused toframework sequences derived from the human germline VH sequence VH2-70to create a polynucleotide encoding a V_(H) designated Hu3G8VH. Thepolynucleotide was generated by an overlapping PCR procedure. In a firststep, using the primers and strategy shown below and the mouse 3G8 V_(H)polynucleotide (SEQ ID NO: 1) as template.

Primer Length Sequence Seq ID No: SJ29f 62 ccg cga att ctG GCC AGG TTACCC TGA GAG 7 AGT CTG GCC CTG CGC TGG TGA AGC CCA CAC AG SJ30f 80 GCGCTG GTG AAG CCC ACA CAG ACC CTC ACA 8 CTG ACT TGT ACC TTC TCT GGG TTTTCA CTG AGC ACT TCT GGT ATG GGT GT SJ31f 42 TGG ATT CGT CAG CCT CCC GGGAAG GCT CTA 9 GAG TGG CTG GCA SJ32r 42 TGC CAG CCA CTC TAG AGC CTT CCCGGG AGG 10 CTG ACG AAT CCA SJ33f 72 GTC CTC ACA ATG ACC AAC ATG GAC CCTGTG 11 GAT ACT GCC ACA TAC TAC TGT GCT CGG ATA AAC CCC GCC TGG SJ34r 51CAT GTT GGT CAT TGT GAG GAC TAC CTG GTT 12 TTT GGA GGT ATC CTT GGA GATSJ35r 37 GGC TGA GCT CAC AGT GAC CAG AGT CCC TTG 13 GCC CCA G SJ37f 27GTG TAG GCT GGA TTC GTC AGC CTC CCG 14 SJ38r 33 GAC GAA TCC AGC CTA CACCCA TAC CAG AAG 15 TGC

The resulting fragment was digested with EcoRI and SacI and cloned intopUC18. After sequencing, one plasmid was selected for a final round ofoverlapping PCR to correct a deletion which occurred during the secondPCR step. The resulting polynucleotide had the sequence:

SEQ ID NO: 16 {hu3G8VH}CAGGTTACCCTGAGAGAGTCTGGCCCTGCGCTGGTGAAGCCCACACAGACCCTCACACTGACTTGTACCTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTCCCGGGAAGGCTCTAGAGTGGCTGGCACACATTTGGTGGGATGATGACAAGCGCTATAATCCAGCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCAAAAACCAGGTAGTCCTCACAATGACCAACATGGACCCTGTGGATACTGCCACATACTACTGTGCTCGGATAAACCCCGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTGAG CTCA

The Hu3G8V_(H) sequence was then combined with segments coding for asecretion signal sequence (as described above; lower case underline) andcDNA for the human Cγ1 constant region (lower case). The resultingpolynucleotide had the sequence:

SEQ ID NO: 17 {hu3G8VH-1}gctagcgtttaaacttaagcttgttgactagtgagatcacagttctctctacagttactgagcacacaggacctcaccatgggatggagctgtatcatcctcttcttggtagcaacagctacaggtaaggggctcacagtagcaggcttgaggtctggacatatatatgggtgacaatgacatccactttgcctttctctccacaggtgtccactccCAGGTTACCCTCAGAGAGTCTGGCCCTGCGCTGGTGAAGCCCACACAGACCCTCACACTGACTTGTACCTTCTCTGCGTTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTCCCGGGAAGCCTCTAGAGTGGCTGGCACACATTTCGTGGGATGATGACAAGCGCTATAATCCAGCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCAAAAACCAGGTAGTCCTCACAATGACCAACATGGACCCTGTGGATACTGCCACATATCTACTGTGCTCGGATAAACCCCGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTGAGCTCAgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactccggggggaccgtcagtcttcctcttcccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcctctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagtgcggccgcgaattc

For expression in mammalian cells (HEK-293), the Hu3G8VH-1 sequence wascloned into the pCI-Neo polylinker at the NheI-EcoRI sites, followingintervening cloning into pUC and pcDNA3.1.

B) Humanized Light Chain

CDR encoding sequences from the mouse 3G8 V_(L) clone were fused toframework sequences derived from the human B3 germline V-κ gene. Thepolynucleotide was generated by an overlapping PCR procedure using theprimers and strategy shown below and the mouse 3G8 V_(L) polynucleotide(SEQ ID NO: 2) as template.

Primer Length Sequence Seq ID No: H023 63ACTCTTTGGCTGTGTCTCTAGGGGAGAGGGCCACCATCAACTG 18 CAAGGCCAGCCAAAGTGTTG H02466 CTCTCCACAGGTGTCCACTCCGACATCGTGATGACCCAATCTC 19CAGACTCTTTGGCTGTGTCTCTA H025 71GGTGAGGGTGAAGTCTGTCCCAGACCCACTGCCACTAAACCTG 20TCTGGGACCCCAGATTCTAGATTGGATG H026 67TGACAGTAATAAACTGCCACATCCTCAGCCTGCAGGCTGCTGA 21 TGGTGAGGGTGAAGTCTGTCCCAGH027 71 gcggcAAGCTTGGTCCCCTGTCCGAACGTGTACGGATCCTCAT 22TACTTTGCTGACAGTAATAAACTGCCAC H009 30 CAT GTT GGT CAT TGT GAG GAC TAC CTGGTT TTT 12 GGA GGT ATC CCT GGA GAT SJ35r 37 GGC TGA GCT CAC AGT GAC CAGAGT CCC TTG GCC 13 CCA G SJ37f 27 GTG TAG GCT GGA TTC GTC AGC CTC CCG 14SJ38r 33 CGAGCTAGCTGAGATCACAGTTCTCTCTAC 23

The resulting polynucleotide had the sequence

SEQ ID NO: 25 {hu3G8VL}GACACTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTTTGATGGTCATAGTTTTATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATACTACATCCAATCTAGAATCTGGGATCCCAGCCAGCTTTAGTGCCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATACTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGAGGGGGGACCAAGCTTGAGATCAAA

The Hu3G8 V_(L) gene segment was combined with a signal sequence (asdescribed above, lower case, underline) and a human C-κ constant region(lower case) cDNA using standard techniques resulting in a product withthe sequence below:

SEQ ID NO: 26 {hu3G8VL-1}gctagctgagatcacagttctctctacagttactgagcacacaggacctcaccatgggatggagctgtatcatcctcttcttggtagcaacagctacaggtaaggggctcacagtagcaggcttgaggtctggacatatatatgggtgacaatgacatccactttgcctttctctccacaggtgtccactccGACACTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGCCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTTTGATGCTGATAGTTTTATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATACTACATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGCCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGCAGGAGGATACTGCAACCTATTACTGTCAGCAAAGTAATGAAATCCGTACACGTTCGGAGGGGGGACCAAGCTTGAGATCAAAcgaactgtggctgcaccatcggtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttagttctagagtcgactctagaggatccccgggtaccgagctcgaattc

This construct was inserted into pCI-Neo for expression in mammaliancells.

Example 3 Variant CD16A Binding Proteins

Additional expression constructs were made in which sequence changeswere introduced in the V_(L) or V_(H) domains by site directedmutagenesis. A typical mutagenesis reaction contained 10 ng plasmid DNA(isolated from a methylation competent strain of E. coli), 125 ng eachof a forward and reverse primer, each containing the mutation ofinterest, reaction buffer, and dNTPs in 0.05 ml volume. 2.5 units ofPfuTurbo DNA polymerase (Stratagene) was added and the reaction wassubjected to 15 cycles of 95°, 30 sec; 55°, 1 min; 68°, 12 min. Theproduct of the PCR was then digested with DpnI endonuclease and therestricted DNA was used to transform E. coli strain XL-10 gold. BecauseDpnI only digests methylated DNA it will digest the parental,non-mutated, plasmid leaving the newly synthesized non-methylatedproduct, containing the mutation of interest, as the predominantspecies.

The sequences of the variant V_(H) domains are shown in Table 4. Thesequences of the variant V_(L) domains are shown in Table 5.

Example 4 Expression in Mammalian Cells

Various combinations of heavy and light chain expression plasmids (e.g.,comprising the chimeric, humanized and variant V_(L) and V_(H) domainsfused to human Cγ1 and Cκ constant domains as described above) wereco-transfected into HEK-293 cells for transient expression ofrecombinant tetrameric antibodies (i.e., comprising 2 heavy chains and 2light chains), sometimes referred to herein as “recombinant antibodies.”Transfection was carried out using Lipofectamine-2000 (Invitrogen) in 6well plates according to the manufacturer's instructions.

Recombinant antibodies were prepared by cotransfection of a heavy chainexpression plasmid (i.e., encoding a heavy chain comprising a V_(H) andconstant domains) and light chain expression plasmids (i.e., encoding alight chain comprising a V_(L) and constant domains) together intoHEK-293 cells for transient expression of recombinant antibodies.

Hu3G8VH variants listed in Table 4 were coexpressed with the hu3G8VL-1light chain. For reference, most assays included (i) recombinantantibodies produced by coexpression of ch3G8VH and ch3G8VL(“ch3G8VH/ch3G8VL”) and (ii) recombinant antibodies produced bycoexpression of hu3G8VH-1 and hu3G8VL-1 (“hu3G8VH-1/hu3G8VL-1”).

Hu3G8VL variants listed in Table 5 were coexpressed with the ch3G8VHheavy chain. For reference, most assays included (i) recombinantantibodies produced by coexpression of ch3G8VH and ch3G8VL(“ch3G8VH/ch3G8VL”) and (ii) recombinant antibodies produced bycoexpression of ch3G8VH and hu3G8VL-1 (“ch3G8VH/hu3G8VL-1”).

After three days, the levels of recombinant antibodies in theconditioned media were determined by ELISA, and the recombinantantibodies were analyzed by ELISA for binding to captured sCD16A asdescribed in Examples 5. Selected antibodies were assayed for cellbinding to cells expressing the extracellular domain of CD16A, as shownin Example 6.

Example 5 ELISA Determination of Binding to CD16A

Sandwich ELISA was performed to detect binding of antibodies to asoluble form of CD16A.

Soluble Human CD16A

A soluble form of human CD16A was expressed from HEK-293 cells using apcDNA3.1-derived expression vector containing the CD16A gene truncatedjust prior to the transmembrane region. To create the vector, cDNAencoding CD16A was amplified using the primers 3A_(left)[gttggatcctccaactgctctgctacttctagttt] (SEQ ID NO:27) and 3A_(right)[gaaaagcttaaagaatgatgagatggttgacact] (SEQ ID NO:28) digested with BamHIand HindIII, and cloned into the vector pcDNA3.1 (Novagen) at theBam/HindIII site of the polylinker. The construct was used totransiently transfect HEK-293 cells. For some assays, the secretedproduct was purified from conditioned medium using affinitychromatography on a human IgG Sepharose column. In some assays, theamount of sCD16A in conditioned medium was quantitated and unpurifiedsCD16A was used. Purification was not required since the ELISA captureantibody (LNK16 mAb) specifically bound the antigen, allowing removal ofcontaminants in washing steps.

The amino acid sequence of the sCD16 construct is shown below. (Thesignal sequence, underlined, is cleaved off during expression. Note thelast seven residues are derived from the vector pcDNA3.1 rather thanfrom the CD16A gene):

(SEQ ID NO: 29) MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTIS SFFKLAAARV

ELISA Format

Plates were first coated with 100 ng/well of the anti-CD16 mAb LNK-16(Advanced ImmunoChemical, Long Beach Calif.; see 5th Human LymphocyteDifferentiation Antigens Workshop) in carbonate buffer at roomtemperature for 2 hrs. Any anti-sCD16A antibody that does not blockbinding by 3G8 can be used. After blocking for 30 minutes withPBS-T-BSA, sCD16A conditioned medium was added at a dilution of 1/10 andincubated at room temperature for 16 hrs. Alternatively, when purifiedsCD16 was used, it was diluted to a concentration of 50 ng/ml inPBS-T-BSA. 0.05 ml was added to each well and incubated for at least 2hrs at room temperature.

The plate washed and dilutions of recombinant antibodies starting at 0.5μg/ml in PBS-T-BSA were then added and incubated for 1 hr at room temp.Binding of recombinant antibodies to the captured sCD16A was thenmeasured using an anti-human IgG-HRP conjugate and TMB substrate. Afterstopping color development using dilute sulfuric acid, the plate wasread at 450 nM.

Results of Binding Assays

This example shows that the binding properties of humanized anti-CD16Aantibodies for binding to CD16A are the same or similar to theproperties of the chimeric 3G8 antibody.

Based on the comparative binding studies, the recombinant antibodieswere classified as binding with high, intermediate, or low affinity.Antibodies with high and intermediate binding affinity are discussedabove in section 4. The recombinant antibodies with a V_(H) domain ofhu3G8VH-9, 10, 11, 13, 15, 21, 38, 39, or 41 showed little or no bindingto sCD16A. From these data it appears certain substitutions (orcombinations of substitutions) are generally detrimental to binding. Forexample, substitution of tyrosine or aspartic acid at V_(H) position 52(i.e., 52Y and 52D) or threonine at position 94 (94T) are detrimental tobinding. Similarly, the combination leucine at position 50 with asparticacid at position 54 (50L+54N) is detrimental to binding, as is thecombination arginine at 94 and aspartic acid at 101 (94R+101D). However,aspartic acid at 101 is tolerated when position 94 is glutamine, lysine,histidine or alanine (but not arginine). Further 34V+94R+101D hasintermediate activity. This indicates a relationship between positions34, 94 and 101 in maintaining high affinity binding, and suggests that34V may be an especially important residue. Likewise, recombinantantibodies with a V_(L) domain of hu3G8VL-6, 7, 8, 9, 11, 12, 13, and 14showed little or no binding to sCD16A. From these data it appearscertain substitutions (or combinations of substitutions) are generallydetrimental to binding. For example, substitution of alanine at position34 (34A) or tyrosine at position 92 (92Y) is generally detrimental tobinding.

Results of an exemplary binding assay are shown in FIG. 1.

Example 6 Antibody Binding to Cells Expressing CD16A

The ability of selected humanized antibodies to bind to CD16A expressedby CHO-K1 cells as assayed by direct binding competition assays.

CHO-K1 cells expressing extracellular domain of FcRIIIa fused to thetransmembrane and intracellular domain of FcRIIb were used for cellbinding assays. Cells were plated at 40,000 cells per well in 96 wellflat bottom tissue culture plates (FALCON MICROTEST Tissue Cultureplate, 96 well) and incubated at 37° C. CO₂ incubator for approximately24 hr. The plate was then gently washed three times with 25 mM Hepes, 75μM EDTA, 11.5 mM KCl, 115 mM NaCl, 6 mM MgSO4, 1.8 mM CaCl2, 0.25% BSA(binding buffer).

For indirect binding assays, 100 μl of a serial dilution of anti-CD16Mab (final concentration: 1 μg/ml, 0.5, 0.25, 0.125, 0.0625, 0.03,0.015, 0 μg/ml) was then added to wells in binding buffer. The plate wasthen incubated at 23° C. for 1 hr and washed three times with bindingbuffer. 50 μl/well of Europium (EU)-labeled-anti-human-IgG (100 ng/ml)was then added to each well and the plate was incubated at 23° C. for 30minutes then washed three times with binding buffer. Finally, 100 μlDelfia enhancement solution (PerkinElmer/Wallac) was added. Afterincubating with shaking for 15 minutes, the plate was read for timeresolved fluorescence (excitation 340 nm; emission 615 nm) in a Victor2instrument (PerkinElmer/Wallac). The results of the assay are shown inFIG. 2.

The CHO-K1 cells described above were also used in competition assays.After washing with binding buffer as described above, varying amounts ofpurified unlabeled Mab (1.2-75 nM final concentration) were mixed with afixed concentration of Eu—Ch3G8-N297Q (final concentration 2.5 nM). Theplate was then incubated at 23° C. for 1 hr and washed three times withbinding buffer. 100 μl Delfia enhancement solution (PerkinElmer/Wallac)was the added and after incubating with shaking for 15 minutes, theplate was read for time resolved fluorescence (excitation 340 nm;emission 615 nm) in a Victor2 instrument (PerkinElmer/Wallac). Theresults of the assay are shown in FIG. 3.

These assays demonstrate that the humanized anti CD16A monoclonalantibodies bind with high affinity to CD16A on the surface oftransfected cells. Hu3G8-22.1-N297Q binds to CD16A bearing cells withhigher affinity than Ch3G8-N297Q.

Example 7 Inhibition of Binding of sCD16A to Immune Complexes

Assay of 4-4-20 Binding to FITC-BSA

The binding of ch4-4-20 or ch4-4-20 (D265A) to FITC-BSA was assessed byELISA. (Ch4-4-20 is identical to Ch3G8 except that it contains therespective VH and VL regions of 4-4-20 instead of those of 3G8. Thus itretains high affinity and specificity for the hapten fluorescein. 4-4-20is described in Bedzyk et al., 1989, J Biol Chem 264:1565-9.) FITC-BSA(1 μg/ml-50 ng/well) was coated onto Nunc maxisorb immunoplates incarbonate buffer and allowed to bind for approximately 16 hr. Followingblocking with BSA, dilutions of ch4-4-20 were added to the wells andallowed to bind for 1 hr at RT. After washing out unbound Mab,HRP-conjugated goat anti-human Ig secondary was added. One hour laterthe secondary antibody was removed, washed and developed with TMBsubstrate. Following addition of an acidic stop solution the plate wasread at 450 nm. Both ch4-4-20 and ch4-4-20(D265A) bound to the FITC-BSAwith high affinity (data not shown).

Assay of sFcR Binding to ch4-4-20/FITC-BSA Immune Complexes

The same format was used to assay binding of sFcRs to immune complexes(IC) formed on the ELISA plate between ch4-4-20 and FITC-BSA. In thiscase we have used either biotinylated sFcR or biotinylated anti-human G2Mab as a secondary reagent, followed by streptavidin-HRP detection.

Inhibition of sFcR Binding to IC by Murine, Chimeric and Humanized 3G8

The concentrations of ch4-4-20 and sFcR were fixed to give approximately90 percent maximal signal in the assay. sCD16A was premixed with serialdilutions of murine, chimeric or humanized 3G8 and incubated for onehour prior to adding to the plate containing the immune complex. Serialdilutions of humanized or chimeric 3G8 were incubated withsCD16A-G2-biotin for one hour. The mixtures were then added to ELISAwells containing an immune complex between a human IgG1 chimeric form ofthe anti-fluorescein Mab 4-4-20 and FITC-BSA. After one hour, binding ofthe soluble receptor to the IC was detected using streptavidin-HRPconjugate and TMB development. The results are shown in FIG. 4. Thisassay indicates that humanized anti-CD16A antibodies are potentinhibitors of CD16A binding to IgG in immune complexes.

Example 8 Analysis of Anti-CD16A Monoclonal Antibody Panel

A panel of hybridomas was generated following immunizing and boostingmice with sCD16A using standard methods. Eight 96-well plates werescreened by ELISA for binding activity on plates coated directly withsCD16A. Ninety-three of these gave a positive signal and were expandedfurther. Of these, 37 were positive for binding to human blood cells byFACS. These supernatants were then analyzed for their ability to blockthe interaction of CD16A with immune complexes and for the similarity ofthe binding site (epitope) to that of 3G8. Assays included capture ELISAusing chimeric 3G8 down and inhibition of immune complex binding tosRIIIa-Ig. Based on these assays antibodies with binding and inhibitoryproperties similar to 3G8 were isolated, as well as Mabs with bindingand/or inhibitory properties distinct from 3G8.

DJ130c (DAKO) and 3G8 were used as controls in the assays. Mab DJ130c isa commercially available Mab which binds CD16 at an epitope distinctfrom 3G8 (Tamm and Schmidt). This Mab does not block FcRIIIa-immunecomplex binding (Tamm and Schmidt). In an ELISA-based inhibition assay,DJ130c enhances rather than inhibits binding.

The data presented in Table 3 indicate that the panel containsantibodies which bind to the same epitope as Ch3G8 and block sCD16Abinding to immune complexes. The panel of Mabs also contains antibodieswhich do not bind to the same epitope as Ch3G8. Most of these latterantibodies do not block the interaction of sCD16a with IgG in immunecomplexes.

TABLE 3 Effect of sCDa Binding to Immune Compplexes Assay ResultInhibition Enhancement No Effect Binding to Positive 2 5 (+DJ-130c) 17sCD16 Negative 11 (+3G8) 0 2 Captured by ch3G8

Example 9 Induction of Platelet Depletion In Vivo

The in vivo activity of a CD16A binding protein for blocking humanFc-FcγRIII interactions induced by autoantibodies can be evaluated usinganimal models of autoimmune diseases. One suitable model is the “passivemouse model” of ITP and the anti-platelet mAb 6A6 (see, Oyaizu et al.,1988, J Exp. Med. 167:2017-22; Mizutani et al, 1993, Blood 82:837-44).6A6 is an IgG2a isotype mAb derived from a NZW.times.BSXB F1 individual.Administration of 6A6 depletes platelets in muFcγRIII −/−, huFcγRIIIAtransgenic mice but not in muFcγRIII −/− mice without the humantransgene. See Samuelsson et al., 2001, Science 291:484-86. Otheranti-platelet monoclonal antibodies can be used in place of 6A6 in themodel. Alternatively, a polyclonal anti-platelet antibody can be used.

CD16A binding proteins that confer the greatest degree of protectionfrom platelet depletion can be identified by administrating CD16Abinding proteins to a muFcγRIII −/−, huFcγRIIIA transgenic mouse andmeasuring any reduction in mAb 6A6 induced platelet depletion.

A related assay can be carried out using a chimeric human IgG₁ κchimeric derivative of 6A6 in place of the mouse mAb in the protocolprovided above, so that the depleting mAb had a human isotype. Toconduct this assay, a chimeric 6A6 monoclonal antibody (ch6A6) wasprepared by fusing the cDNA segments encoding the murine anti-plateletmonoclonal antibody 6A6 V_(H) and V_(L) regions to the human Cγ1 and CκcDNA segments, respectively. The resulting genes were co-expressed in293 cells and chimeric 6A6 was purified by protein A affinitychromatography followed by size exclusion chromatography.

To demonstrate that the chimeric 6A6 antibody induces plateletdepletion, to and ch6A6 was administered to muFcγRIII^(−/−), huFcγRIIIAtransgenic mice. The ch6A6 was administered to each animal either i.v.or intraperitoneally (i.p.) (0.1 μg/g). Animals were bled 2 hrs, 5 hrs,24 hrs and 48 hrs after administration of ch6A6, and plasma plateletcounts were determined using a Coulter Z2 particle counter and sizeanalyzer equipped with a 70 μm aperture. Particles between 1.5 and 4 μmin size (corresponding to platelets) were counted and the data wereanalyzed by plotting the platelet count versus time for eachconcentration.

Two hours after injection of 0.1 μg/g ch6A6 i.p., approximately 75% ofthe platelets were depleted. The number of platelets remained low for 5hours after ch6A6 injection then progressively increased to return tonormal 72 hours after ch6A6 injection.

Two hours after injection of 0.1 μg/g ch6A6 i.v., approximately 60% ofthe platelets were depleted. The number of platelets remained low for 6hours after ch6A6 injection then progressively increased to return tonormal 48 hours after ch6A6 injection.

Example 10 Analysis of the Ability of CD16 Binding Antibodies to ProtectMice from Platelet Depletion

The ability of CD16A binding proteins to reduce platelet depletion inexperimental ITP can be assayed as described below. CD16A bindingproteins were administered intravenously (i.v.) to groups ofmuFcγRIII−/−, huFcγRIIIA transgenic mice at concentrations of 0.5, 1, 2or 5 μg/g in phosphate buffered saline (PBS). Controls were PBS alone oran irrelevant human IgG1 (negative control) or human intravenousimmunoglobulin (IVIG; positive control). One hour after administrationof the CD16A binding protein or control, ITP was induced byadministering 0.1 μg/g ch6A6 to each animal either intravenously orintraperitoneally. Animals were bled 2 hrs, 5 hrs, 24 hrs and 48 hrsafter administration of ch6A6. Plasma platelet counts were determinedusing the Coulter Z2 particle counter and size analyzer as describedabove and the data were analyzed by plotting the platelet count versustime for each concentration of administered binding protein.

When muFcγRIII^(−/−), huFcγRIIIA transgenic mice were injected withmurine 3G8 (0.5 μg/g) one hour before i.p. injection of ch6A6, 33% ofthe platelets were depleted at the 2 hours time point (FIG. 5). Thenumber of platelets then progressively increased to return to normal 24hours after ch6A6 injection. When muFcγRIII^(−/−), huFcγRIIIA transgenicmice were injected with murine 3G8 (0.5 μg/g) one hour before i.v.injection of ch6A6, 30% of the platelets were depleted at the 2 hourstime point (FIG. 6). The number of platelets then rapidly increased toreturn to normal 5 hours after ch6A6 injection.

These results were similar to the protection seen when human IVIG isadministered. When muFcγRIII^(−/−), huFcγRIIIA transgenic mice wereinjected with human IVIG (1 mg/g) one hour before i.p. injection ofch6A6, 33% of the platelets were depleted at the 2 hours time point(FIG. 5). The number of platelets then progressively increased to returnto normal 24 hours after ch6A6 injection. When muFcγRIII^(−/−),huFcγRIIIA transgenic mice were injected with human IVIG (1 mg/g) onehour before i.v. injection of ch6A6, 20% of the platelets were depletedat the 2 hours time point (FIG. 6). The number of platelets then rapidlyincreased to return to normal 5 hours after ch6A6 injection.

The results shown in FIGS. 5 and 6 show that m3G8 protects mice fromch6A6-mediated platelet depletion, and that the level of protection wassimilar to the protection conferred by IVIG.

Preparations of recombinant mouse 3G8 produced in HEK-293 cells,chimeric 3G8 with human IgG1 or IgG2 constant domains (ch3G8-γ1 producedin HEK-293 and CHO-K1 cells, and ch3G8-γ2 produced in HEK-293 cells),and a ch3G8-γ1 variant (ch3G8-γ1 D265A) did not provide significantprotection in this experiment. When muFcγRIII^(−/−), huFcγRIIIAtransgenic mice were injected with ch3G8γ1 or γ2 (0.5 μg/g) one hourbefore i.p. injection of 6A6, approximately 60% of the platelets weredepleted at the 5 hour time point (FIG. 7). The number of platelets thenprogressively returned to normal. Although depletion was not as severeas in mice that received no anti-CD16A binding protein, these chimericantibodies provided significantly less protection, if any, than murine3G8. A ch3G8 variant in which aspartic acid 265 was changed to alanineshowed similar results. Interestingly, as is shown in Example 11,modification of the ch3G8 to produce an aglycosylated variant increasedthe protective effect of the antibody.

Example 11 Ch3G8 N2970 Protects Mice from ch6A6-Mediated PlateletDepletion

An aglycosylated version of ch3G8-γ1 was prepared by mutating theexpression polynucleotide encoding ch3G8-γ1 so that residue 297 waschanged from asparagine (N) to glutamine acid (Q), and expressing theencoded antibody. Residue 297 lies in an N-linked glycosylation site,and this mutation prevents glycosylation of the Fc domain at this site.This aglycosylated antibody, ch3G8 N297Q, was produced in HEK-293 cellsas described for ch3G8-γ1 (see Example 4, supra). The ability ofch3G8-N297Q to protect against ch6A6-mediated platelet depletion wastested using the protocol described above.

When muFcγRIII^(−/−), huFcγRIIIA transgenic mice were injected with 1μg/g of the aglycosyl form of ch3G8 (ch3G8 N297Q) one hour before i.p.injection of ch6A6, approximately 75% of the platelets were depleted atthe 2-hour time point (FIG. 8). Platelet levels increased faster than inthe absence of ch3G8 N297Q, and returned to normal by 24 hours afterch6A6 injection.

When muFcγRIII^(−/−), huFcγRIIIA transgenic mice were injected with 1μg/g ch3G8 N297Q one hour before i.v. injection of ch6A6, approximately60% of the platelets were depleted at the 2 hours time point (FIG. 9).Platelet levels increased faster than in the absence of ch3G8 N297Q, andreturned to normal by 48 hours after ch6A6 injection.

When muFcγRIII^(−/−), huFcγRIIIA transgenic mice were injected withch3G8 N297Q (2 μg/g) one hour before i.v. injection of ch6A6, only 40%of the platelets were depleted at the 2 hours time point (FIG. 9).Platelet levels increased faster than in the absence of ch3G8 N297Q, andreturned to normal by 5 hours after ch6A6 injection.

Thus, ch3G8-N297Q was consistently able to significantly improveplatelet counts. Binding of 3G8 to human CD16 on effector cells blocksthe ability of CD16 to interact with immune complexes and triggereffector functions such as ADCC or phagocytosis. Chimeric and mouse 3G8molecules have similar ability to bind CD16 and are similar in theirability to inhibit the binding of sCD16 to immune complexes in vitro.Without intending to be bound by a particular mechanism, the binding(and thus) the blocking activity of the mAb is thought to be confined tothe Fab portion of the antibody and blocking of huCD16 is believed to bethe mechanism of protection in the transgenic mouse ITP model. The dataabove suggest that the glycosylation state of the Fc domain can affectthe in vivo protective capacity of anti-CD16A antibodies. Ablation of Fcdomain glycosylation (e.g., with D265A or N297Q mutations, or by using ahuman gamma2 Fc domain) reduces or eliminates Fc binding to FcR. In thecase of the aglycosyl (N297Q) variant, complement fixation is alsoabolished.

Example 12 Neutrophil Levels Following Administration of Aglycosyl CD16ABinding Proteins

The effect of an aglycosylated CD16A binding protein on neutrophillevels was tested and compared to that of glycosylated CD16A bindingproteins. CD16A binding proteins, or the controls such as irrelevanthuman IgG1 (negative control) or murine RB6-8C5 (positive control), wereadministered to groups of muFcγRII^(−/−), huFcγRIIIB transgenic mice ata concentration of 5 μg/g in phosphate buffered saline (PBS). Anothernegative control was administered PBS alone. Twenty four hours later,mice were euthanized and blood, spleen and bone marrow are collected.Neutrophils were analyzed by FACS. Staining experiments were performedin RPMI containing 3% FCS. Murine cells were stained usingFITC-conjugated 3G8 (PharMingen) and R-PE-conjugated RB6-8C5(PharMingen). Samples were analyzed by flow-cytometry using aFACSCalibur (Becton Dickinson).

Intraperitoneal injection of 5 μg/g ch3G8 (prepared as described above)resulted in murine neutrophil depletion in the blood and spleen (FIG.10; upper right quadrant). Similar results were seen followingadministration of murine 3G8 (results not shown). In the bone marrow ofch3G8 treated animals, neutrophils stained weakly for CD16, which couldindicate receptor occupancy by the chimeric antibody or shedding (FIG.10; see shift from the upper right quadrant to the upper left quadrant).In contrast, intraperitoneal injection of 5 μg/g ch3G8 N297Q did notresult in murine neutrophil depletion in the blood, spleen or bonemarrow (FIG. 10). In additional experiments, humanized glycosylated 3G8antibodies showed substantially more depletion of circulating bloodneutrophils compared to aglycosylated forms of the same antibodies.

Example 13 Autoimmune Hemolytic Anemia Model

This example demonstrates that administration of CD16A binding proteinprevents red blood cell depletion in a model of autoimmune hemolyticanemia.

The ability of the Hu3G8-5,1-N297Q monoclonal antibody to preventantibody-dependent red blood cell depletion in muFcRIII^(−/−),huFcRIIIa+ mice was evaluated. Hu3G8-5,1-N297Q is an aglycosy antibodywith the heavy chain Hu3G8VH-5 and the light chain Hu3G8VH-1 and theindicated substitution of asparagine 297. Mice were bled on day 0 andRBC levels were determined using a Coulter Z2 particle analyzer. Thenext day groups of 3 animals each were then injected intravenously witheither 0.5 mg/kg Hu3G8-5.1-N297Q or PBS. One group of mice did notreceive any compound. One hour later, RBC depletion was induced in thefirst two groups by administering mouse anti-RBC IgG2a Mab 34-3C to eachanimal intraperitoneally (i.p.) (2.5 mg/kg). Animals were bled 2 hrs, 5hrs, 24 hrs and 48 hrs after administration of 34-3C and RBC counts weredetermined. Data was analyzed by plotting RBC count versus. The data,depicted in FIG. 11, demonstrate the ability of Hu3G8-5.1-N297Q toprevent RBC depletion in this model.

Example 14 Inhibition of Antibody-Dependent Cellular Cytotoxicity (ADCC)

This example demonstrates that humanized 3G8 variants inhibit ADCC invitro and with an activity similar to that of mouse 3G8.

Methods: The protocol for assessment of antibody dependent cellularcytotoxicity (ADCC) is similar to that previously described in (Ding etal., 1998, Immunity 8:403-11). Briefly, target cells from theHER2-overexpressing breast cancer cell line SK-BR-3 were labeled withthe europium chelate bis(acetoxymethyl)2,2′:6′,2″-terpyridine-6,6″-dicarboxy-late (DELFIA BATDA Reagent, PerkinElmer/Wallac). The labeled target cells were then opsonized (coated)with either chimeric anti-HER2 (ch4D5, 100 ng/ml) or chimericanti-fluorescein (ch4-4-20, 1 μg/ml) antibodies. In the case of theanti-fluorescein antibody, SK-BR-3 cells were coated with thefluorescein hapten prior to antibody opsonization. Peripheral bloodmononuclear cells (PBMC), isolated by Ficoll-Paque (Amersham Pharmacia)gradient centrifugation, were used as effector cells (Effector:Targetratio: ch4D5=(37.5:1) and ch4-4-20=(75:1)). Following a 3.5 hourincubation at 37° C., 5% CO2, cell supernatants were harvested and addedto an acidic europium solution (DELFIA Europium Solution, PerkinElmer/Wallac). The fluorescence of the Europium-TDA chelates formed wasquantitated in a time-resolved fluorometer (Victor2 1420, PerkinElmer/Wallac). Maximal release (MR) and spontaneous release (SR) weredetermined by incubation of target cells with 2% TX-100 and media alone,respectively. Antibody independent cellular cytotoxicity (AICC) wasmeasured by incubation of target and effector cells in the absence ofantibody. Each assay is performed in triplicate. The mean percentagespecific lysis was calculated as: (ADCC−AICC)/(MR−SR).times.100.

Results: Addition of anti-CD16 variants inhibited ADCC mediated throughantibodies directed against the HER2/neu protein (ch4D5) (FIG. 12), orthe hapten, fluorescein (ch4-4-20) (FIG. 13). Inhibition of the ch4D5mediated ADCC was greater than 50% at 300 ng/ml for all 3G8 variantstested while isotype control antibodies had no effect in the assay. Inthe case of the anti-fluorescein antibody, inhibition was approximately50% at concentrations above 1 ug/ml for murine 3G8 (FIG. 13A) andhumanized 3G8 variants (FIG. 13B), while isotype control antibodies andchimeric 3G8 had little effect.

Example 15 Administration of Hu3G8-5,1-N297Q Prevents ImmuneThrombocytopenia (ITP) in huFcRIIa+, huFcRIIIa+Mice

This example shows that that administration of anti-CD16A antibodiesprotects against ITP mediated by CD32A. As in FcγRIII−/−, hCD16A mice,administration of the ch6A6 antibody induces ITP in FcγRIII−/−, hCD32Atransgenic mice. Five hours after injection of 0.1 μg/g ch6A6 i.p.,approximately 80% of the platelets are depleted (not shown). The numberof platelets remained low for 24 hours after ch6A6 injection, and thenprogressively increased to return to normal 48 hours after ch6A6injection. As expected, the i.v. injection of hu3G8-5.1 (0.5 μg/g) onehour prior to ch6A6 injection did not protect FcγRIII^(−/−), hCD32A miceagainst ITP (not shown).

As in single transgenic mice, ch6A6 induces ITP in FcγRIII−/−, hCD16A,hCD32A double transgenic mice. Five hours after injection of 0.1 μg/gch6A6 i.p., approximately 80% of the platelets were depleted (FIG. 14).The number of platelets remained low for 24 hours after ch6A6 injection,and then progressively increased to return to normal 48 hours afterch6A6 injection.

In contrast to FcγRIII−/−, hCD32A mice, FcγRIII−/−, hCD16A, hCD32A micewere protected against ITP by administration of hu3G8-5.1. Completeprotection was observed when 1 μg/g h3G8 5.1 is injected one hour priorto ch6a6 ip injection; and partial protection resulted fromadministration of or 0.75 μgig or 0.5 μg/g of h3G8 5.1 are used. (FIG.14). Thus, the data indicate that although CD32A can mediate ITP, theinjection of 1 μg/g of h3G8 5.1 completely and unexpectedly protectsmice against platelet depletion.

Example 16 Prevention of Platelet Depletion Using Hu3G8-5.1-N297QProduced in CHO-S Cell Line

Hu3G8-5.1-N297Q was produced in a CHO-S cell line. The ability of thisantibody to protect against ITP in FcγRIII−/−, hCD16A single transgenicmice was determined using the procedure described in Example 13. As isshown in FIG. 15, administration of 0.5 mg/kg or more Hu3G8-5,1-N297Qproduced in CHO-S cells one hour prior to ch6A6 i.p. injectioncompletely protects mice against ITP.

Example 17 Therapeutic Effect of Aglycosylated Humanized Antibodies

ITP was induced in mice as described above, by i.p. injection of 0.1ug/g ch6A6 at time 0. Two hours later, the number of platelets in theplasma was determined to confirm the presence of ITP. Three hours afteri.p. injection of ch6A6, mice were injected i.v. with hu3G8-5.1-N297Q atdifferent concentration (arrow). The results (FIG. 16A) indicate thatthe number of platelets rapidly returns to normal after Hu3G8-5.1-N297Qinjection whereas the number of platelets remains low in non-treatedmice. These results demonstrate that administration of thehu3G8-5.1-N297Q antibody can be used to cure ITP in the mouse model.

In this experiment, ITP was induced by i.p. injection of 0.1 ug/g ch6A6at time 0. Two hours later, the number of platelets in the plasma wasdetermined to confirm the presence of ITP. Three hours after i.p.injection of ch6A6, mice were injected i.v. with hu3G8-22.1-N297Q orhu3G8-22.43-N297Q at 0.5 ug/g (arrow). The results indicate that thenumber of platelets rapidly returns to normal after Hu3G8-22.1-N297Qinjection whereas the number of platelets remains low in non-treatedmice and in mice treated with Hu3G8-22.43-N297Q (FIG. 16B). These dataindicate that hu3G8-22.1-N297Q can be used to cure ITP in the mousemodel.

Example 18 Therapeutic Effect of Hu3G8-22.1-N2970 in AHA inmuFcγRIII−/−, huFcγRIIIA Transgenic Mice

In this experiment, AHA was induced by i.p. injection of 50 ug mouseanti-RBC IgG2a Mab 34-3C at day 0. On day 1, the number of RBC in theblood was determined to confirm the presence of AHA. Two hours later,mice were injected i.v. with Hu3G8-22.1-N297Q at various concentrations(arrow). The results indicate that the number of RBC remained stableafter Hu3G8-22.1-N297Q injection whereas the number of RBC continued todrop in non-treated mice (FIG. 17). The optimal concentration ofHu3G8-22.1-N297Q is 0.5 ug/g. The number of RBC returned to normal inall mice at day 7. Control mice were bled every day but not injected inorder to determine the effect of repeated bleedings on the number ofRBC. These results in the mouse model indicate that Hu3G8-22.1-N297Q canbe used to cure AHA. Hu3G8-22.1-N297Q prevents further RBC depletion byautoantibodies and therefore protects mice against anemia.

Table 4

TABLE 4A* V_(H) SEQUENCES FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 3G8VH A A A A AA A Ch3G8VH A A A A A A B HxC B A B A A A B CxH A A A A B A B Hu3G8VH-1B A B A B A B Hu3G8VH-2 C A B A B A B Hu3G8VH-3 D A B A B A B Hu3G8VH-4B A B A C B B Hu3G8VH-5 B A B A C A B Hu3G8VH-6 B B B A B B B Hu3G8VH-7B B B A B A B Hu3G8VH-8 B A B A B C B Hu3G8VH-9 B A B B B B B Hu3G8VH-10B A B A B B B Hu3G8VH-11 B A B B B A B Hu3G8VH-12 B A B C B A BHu3G8VH-13 B A B D B A B Hu3G8VH-14 B A B E B A B Hu3G8VH-15 B A B A D AB Hu3G8VH-16 B A B A E A B Hu3G8VH-17 B A B A F A B Hu3G8VH-18 B A B A GA B Hu3G8VH-19 B A B A C C B Hu3G8VH-20 B B B C B A B Hu3G8VH-21 B A B AD B B Hu3G8VH-22 B B B C B C B Hu3G8VH-23 B B B C E C B Hu3G8VH-24 B B BC F C B Hu3G8VH-25 B B B C G C B Hu3G8VH-26 B B B C C C B Hu3G8VH-27 B BB C E D B Hu3G8VH-28 B B B C F D B Hu3G8VH-29 B B B C G D B Hu3G8VH-30 BB B C C D B Hu3G8VH-31 E B B C B A B Hu3G8VH-32 E B B H B A B Hu3G8VH-33E B B H B A B Hu3G8VH-34 E B B C B C B Hu3G8VH-35 E B B C C C BHu3G8VH-36 E B B H C D B Hu3G8VH-37 E B B H E C B Hu3G8VH-38 E B B F B AB Hu3G8VH-39 E B B I B A B Hu3G8VH-40 E B B G B A B Hu3G8VH-41 E B B J BA B Hu3G8VH-42 E B B C H A B Hu3G8VH-43 E B B C H C B Hu3G8VH-44 E B B CI D B Hu3G8VH-45 E B B C J D B *Letters in Table 4A refer to sequencesin Tables 4B-H.

TABLE 4B FR1 A B C D E RESIDUE Q Q Q Q Q 1 V V V V I 2 T T T T T 3 L L LL L 4 K R K R K 5 E E E E E 6 S S S S S 7 G G G G G 8 P P P P P 9 G A AA T 10 I L L L L 11 L V V V V 12 Q K K K K 13 P P P P P 14 S T T T T 15Q Q Q Q Q 16 T T T T T 17 L L L L L 18 S T T T T 19 L L L L L 20 T T T TT 21 C C C C C 22 S T T T T 23 F F F F F 24 S S S S S 25 G G G G G 26 FF F F F 27 S S S S S 28 L L L L L 29 R S S R S 30 30 31 32 33 34 Seq IDNo

TABLE 4C CDR1 A B RESIDUE T T 31 S S 32 G G 33 M V 34 G G 35 V V 35A G G35B 35 36 Seq ID No

TABLE 4D FR2 A B RESIDUE W W 36 I I 37 R R 38 Q Q 39 P P 40 S P 41 G G42 K K 43 G A 44 L L 45 E E 46 W W 47 L L 48 A A 49 37 38 Seq ID No.

TABLE 4E CDR2 A B C D E F G H I J RESIDUE H H H H H L H L H L 50 I I I II I I I I I 51 W Y W Y W D F W D W 52 W W W W W W W W W W 53 D N D D N DD D D N 54 D D D D D D D D D D 55 D D D D D D D D D D 56 K K K K K K K KK K 57 R R R R R R R R R R 58 Y Y Y Y Y Y Y Y Y Y 59 N N S N N S S S S S60 P P P P P P P P P P 61 A A S A A S S S S S 62 L L L L L L L L L L 63K K K K K K K K K K 64 S S S S S S S S S S 65 39 40 41 42 43 44 45 46 4748 Seq ID No

TABLE 4F FR3 A B C D E F G H I J RESIDUE R R R R R R R R R R 66 L L L LL L L L L L 67 T T T T T T T T T T 68 I I I I I I I I I I 69 S S S S S SS T T T 70 K K K K K K K K K K 71 D D D D D D D D D D 72 T T T T T T T TT T 73 S S S S S S S S S S 74 S K K K K K K K K K 75 N N N N N N N N N N76. Q Q Q Q Q Q Q Q Q Q 77 V V V V V V V V V V 78 F V V V V V V V V V 79L L L L L L L L L L 80 K T T T T T T T T T 81 I M M M M M M M M M 82 A TT T T T T T T T 82A S N N N N N N N N N 82B V M M M M M M M M M 82C D DD D D D D D D D 83 T P P P P P P P P P 84 A V V V V V V V V V 85 D D D DD D D D D D 86 T T T T T T T T T T 87 A A A A A A A A A A 88 T T T T T TT T T T 89 Y Y Y Y Y Y Y Y Y Y 90 Y Y Y Y Y Y Y Y Y Y 91 C C C C C C C CC C 92 A A A A A A A A A A 93 Q R Q T K A H R H Q 94 49 50 51 52 53 5455 56 57 58 Seq ID No

TABLE 4G CDR3 A B C D RESIDUE I I I I 95 N N N N 96 P P P P 97 A A A A98 W W Y Y 99 F F F F 100 A D A D 101 Y Y Y Y 102 59 60 61 62 Seq ID No

TABLE 4H FR4 A B RESIDUE W W 103 G G 104 Q Q 105 G G 106 T T 107 L L 108V V 109 T T 110 V V 111 S S 112 A S 113 63 64 Seq ID No

Table 5

TABLE 5A* V_(L) SEQUENCES FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 3G8VL A A A A AA A Ch3G8VL A A A A A A A Hu3G8VL-1 B A A A B A B Hu3G8VL-2 B B A A B AB Hu3G8VL-3 B C A A B A B Hu3G8VL-4 B D A A B A B Hu3G8VL-5 B E A A B AB Hu3G8VL-6 B F A A B A B Hu3G8VL-7 B G A A B A B Hu3G8VL-8 B A A B B AB Hu3G8VL-9 B A A C B A B Hu3G8VL-10 B A A D B A B Hu3G8VL-11 B A A E BA B Hu3G8VL-12 B A A F B A B Hu3G8VL-13 B A A G B A B Hu3G8VL-14 B A A AB B B Hu3G8VL-15 B A A A B C B Hu3G8VL-16 B A A A B D B Hu3G8VL-17 B A AA B E B Hu3G8VL-18 B B A D B A B Hu3G8VL-19 B B A D B D B Hu3G8VL-20 B BA D B E B Hu3G8VL-21 B C A D B A B Hu3G8VL-22 B C A D B D B Hu3G8VL-23 BC A D B E B Hu3G8VL-24 B D A D B A B Hu3G8VL-25 B D A D B D B Hu3G8VL-26B D A D B E B Hu3G8VL-27 B E A D B A B Hu3G8VL-28 B E A D B D BHu3G8VL-29 B E A D B E B Hu3G8VL-30 B A A D B D B Hu3G8VL-31 B A A D B EB Hu3G8VL-32 B A A H B A B Hu3G8VL-33 B A A I B A B Hu3G8VL-34 B A A J BA B Hu3G8VL-35 B B A H B D B Hu3G8VL-36 B C A H B D B Hu3G8VL-37 B E A HB D B Hu3G8VL-38 B B A I B D B Hu3G8VL-39 B C A I B D B Hu3G8VL-40 B E AI B D B Hu3G8VL-41 B B A J B D B Hu3G8VL-42 B C A J B D B Hu3G8VL-43 B EA J B D B Hu3G8VL-44 B A A K B A B *Letters in Table 5A refer tosequences in Tables 5B-H.

TABLE 5B FR1 A B RESIDUE D D 1 T I 2 V V 3 L M 4 T T 5 Q Q 6 S S 7 P P 8A D 9 S S 10 L L 11 A A 12 V V 13 S S 14 L L 15 G G 16 Q E 17 R R 18 A A19 T T 20 I I 21 S N 22 C C 23 65 66 Seq ID No

TABLE 5C CDR1 A B C D E F G RESIDUE K R K K K K K 24 A A S A A A A 25 SS S S S S S 26 Q Q Q Q Q Q Q 27 S S S S S S S 27A V V V V V V V 27B D DD D D D D 27C F F F F F F F 27D D D D D D D D 28 G G G G G G G 29 D D DD D D D 30 S S S S S S S 31 F F F Y F F Y 32 M M M M L M L 33 N N N N NA A 34 67 68 69 70 71 72 73 Seq ID No

TABLE 5D FR2 A RESIDUE W 35 Y 36 Q 37 Q 38 K 39 P 40 G 41 Q 42 P 43 P 44K 45 L 46 L 47 I 48 Y 49 74 Seq ID No

TABLE 5E CDR2 A B C D E F G H I J K RESIDUE T D W T D D S S S T T 50 T AA T A A A T T T T 51 S S S S S S S S S S S 52 N N N N N N N N N N S 53 LL L L L L L L L L L 54 E E E E E A Q E Q Q Q 55 S S S T T T S S S S S 5675 76 77 78 79 80 81 82 83 84 85 Seq ID No

TABLE 5F FR3 A B RESIDUE G G 57 I V 58 P P 59 A D 60 R R 61 F F 62 S S63 A G 64 S S 65 G G 66 S S 67 G G 68 T T 69 D D 70 F F 71 T T 72 L L 73N T 74 I I 75 H S 76 P S 77 V L 78 E Q 79 E A 80 E E 81 D D 82 T V 83 AA 84 T V 85 Y Y 86 Y Y 87 C C 88 86 87 Seq ID No

TABLE 5G CDR3 A B C D E RESIDUE Q Q Q Q Q 89 Q Q Q Q Q 90 S S S S S 91 NY Y N N 92 E S E S E 93 D T D D T 94 P P P P P 95 Y Y Y Y Y 96 T T T T T97 88 89 90 91 92 Seq ID No

TABLE 5H FR4 A B RESIDUE F F 98 G G 99 G Q 100 G G 101 T T 102 K K 103 LL 104 E E 105 I I 106 K K 107 93 94 Seq ID No

TABLE 6 Hu3G8VL-1 (SEQ ID NO: 95)CGAGCTAGCTGAGATCACAGTTCTCTCTACAGTTACTGAGCACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCGACATCGTGATGACCCAATCTCCAGACTCTTTGGCTGTGTCTCTAGGGGAGAGGGCCACCATCAACTGCAAGGCCAGCCAAAGTGTTGATTTTGATGGTGATAGTTTTATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATACTACATCCAATCTAGAATCTGGGGTCCCAGACAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAGCCTGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGACACCCCACCAAGCTTGAgATcAAA Hu3G8VL-1 (SEQ ID NO: 96)DIVMTQSPDSLAVSLGERATINCKASQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPY TFGQGTKLEIKHu3G8VL-1K (SEQ ID NO: 97)CGAGCTAGCTGAGATCACAGTTCTCTCTACAGTTACTGAGCACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCGACATCGTGATGACCCAATCTCCAGACTCTTTGGCTGTGTCTCTAGGGGAGAGGGCCACCATCAACTGCAAGGCCAGCCAAAGTGTTGATTTTGATGGTGATAGTTTTATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATACTACATCCAATCTAGAATCTGGGGTCCCAGACAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAGCCTGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAAAGTAATGAGGATCCGTACACGTTCGGACAGGGGACCAAGCTTGAgATcAAACGaACTGTGGCTGCACCATCGGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAA-GGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGTTCTAGAGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTC Hu3GSVL-1K (SEQ ID NO:98) DIVMTQSPDSLAVSLGEEATINCKASQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSNLESGVPDRESGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECHu3G8VL-43 (SEQ ID NO: 99)CGAGCTAGCTGAGATCACAGTTCTCTCTACAGTTACTGAGCACACACGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCGACATCGTGATGACCCAATCTCCAGACTCTTTGGCTGTGTCTCTAGGGGAGAGGGCCACCATCAACTGCAAGtCCAGCCAAAGTGTTGATTTTGATGGTGATAGTTTTATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATACTACATCCAgTCTAGAATCTGGGGTCCCAGACAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAGCCTGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAAAGTAATtcGGATCCGTACACGTTCGGACAGGGGACCAAGCTTGAgATcAAA Hu3G8VL-43 (SEQ ID NO: 100)DIVMTQSPDSLAVSLGERATINCKSSQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSSLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNSDPY TFGQGTKLEIKHu3G8VL-43 + Kappa (SEQ ID NO: 101)CGAGCTAGCTGAGATCACAGTTC-TCTCTACAGTTACTGAGCACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCGACATCGTGATGACCCAATCTCCAGACTCTTTGGCTGTGTCTCTAGGGGAGAGGGCCACCATCAACTGCAAGtCCAGCCAAAGTGTTGATTTTGATGGTGATAGTTTTATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATACTACATCCAgTCTAGAATCTGGGGTCCCAGACAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAGCCTGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAAAGTAATtcGGATCCGTACACGTTCGGACAGGGGACCAAGCTTGAgATcAAACGaACTGTGGCTGCACCATCGGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGTTCTAGAGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTC Hu3G8VL-43K (SEQ IDNO: 102) DIVMTQSPDSLAVSLGERATINCKSSQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSSLESGVPDRFSGSGSGTDFTLTISSLQAWDVAVYYCQQSNSDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECHu3G8VH-1 (SEQ ID NO: 103)GCTAGCgtttaaacttaagcttGTTGACTAGTGAGATCACAGTTCTCTCTACAGTTACTGAGCACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCA-CTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGGTTACCCTGAGAGAGTCTGGCCCTGCGCTGGTGAAGCCCACACAGACCCTCACACTGACTTGTACCTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTCCCGGGAAGGCTCTAGAGTGGCTGGCACACATTTGGTGGGATGATGACAAGCGCTATAATCCAGCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCAAAAACCAGGTAGTCCTCACAATGACCAACATGGACCCTGTGGATACTGCCACATACTACTGTGCTCGGATAAACCCCGCCTGGTTTGCTTACTGGGGCCAAGG GACTCTGGTCACTGTGAGCTCA Hu3G8VH-1 (SEQ ID NO: 104)QVTLRESGPALVKPTQTLTLTCTFSGFSLTSGMGVGWIRQPPGKALEWLAHIWWDDDKRYNPALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTN PAWFAYWGQGTLVTVSSHu3G8VH-1G1 (SEQ ID NO: 105)GCTAGCgtttaaacttaagcttGTTGACTAGTGAGATCACAGTTCTCTCTACAGTTACTGAGCACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGGTTACCCTGAGAGAGTCTGGCCCTGCGCTGGTGAAGCCCACACAGACCCTCACACTGACTTGTACCTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTCCCGGGAAGGCTCTAGAGTGGCTGGCACACATTTGGTGGGATGATGACAAGCGCTATAATCCAGCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCAAAAACCAGGTAGTCCTCACAATGACCAACATGGACCCTGTGGATACTGCCACATACTACTGTGCTCGGATAAACCCCGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTGAGCTCAgcctccaccaagggcccatcggtcttcccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcctccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagtgcggccgcGAATTC Hu3GSVH-1G1 (SEQ ID NO: 107)QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKRYNAPALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARINPAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPFPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTGVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Hu3G8VH-5 (SEQ ID NO: 108)GCTAGCgtttaaacttaagcttGTTGACTAGTGAGATCACAGTTCTCTCTACAGTTACTGAGCACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGGTTACCCTGAGAGAGTCTGGCCCTGCGCTGGTGAAGCCCACACAGACCCTCACACTGACTTGTACCTTGTCTGGGTTTTCACTGAGCACTTCTGGTATGGGGTAGGCTGGATTCGTCAGCCTCCCGGGAAGGCTCTAGAGTGGCTTCAOACATTTGGTGGGATGATGACAAGCGCTATAATCCAGCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCAAAAACCAGGTAGTCCTCACAATGACCAACATGGACCCTGTGGATACTGCCACATACTACTGTGCTCaaATAAACCCCGCCTGGTTTGCTTACTGGGGCCAAGGGAC TCTGGTCACTGTGAGCTCAHu3G8VH-5 (SEQ ID NO: 109)QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKRYNPALKSRLTTSKDTSKNQVVLTNTNMDPVDTATYYCAQT NPAWFAYWGQGTLVTVSSHu3G8VH-5G1Ag (SEQ ID NO: 110)GCTAGCgtttaaacttaagcttGTTGACTAGTGAGATCACAGTTCTCTCTACAGTTACTGAGCACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCOACTCCCAGGTTACCCTGAGAGAGTCTGGCCCTGCGCTGGTGAAGCCCACACAGACCCTCACACTGACTTGTACCTTCTCTGGGTTTTCACTGAGCACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTCCCGGGAAGGCTCTAGAGTGGCTGGCACACATTTGGTGGGATGATGACAAGCGCTATAATCCAGCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCAAAAACCAGGTAGTCCTCACAATGACCAACATGGACCCTGTGGATACTGCCACATACTACTGTGCTCaaATAAACCCCGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTGAGCTCAgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtaccagagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaatgagtgcggccgcGAATTC Hu3G8VH-5G1Ag (SEQ ID NO: 111)QVTLREGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHTWWDDDKRYNPALKSRLTTSKDTSKNQVVLTMTNMDPVDTATYYCAQINPAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDTAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVKDSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Hu3G8VH-22 (SEQ ID NO:112) GCTAGCgtttaaacttaagcttGTTGACTAGTGAGATCACAGTTCTCTCTACAGTTACTGAGCACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGGTTACCCTGAGAGAGTCTGGCCCTGCGCTGGTGAAGCCCACACAGACCCTCACACTGACTTGTACCTTCTCTGGGTTTTCACTGAGCACTTCTGGTgTGGGTGTAGGCTGGATTCGTCAGCCTCCCGGGAAGGCTCTAGAGTGGCTGGCACACATTTGGTGGGATGATGACAAGCGCTATtcTCCAtCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCAAAAACCAGGTAGTCCTCACAATGACCAACATGGACCCTGTGGATACTGCCACATACTACTGTGCTCGGATAAACCCCGCCTacTTTGCTTACTGGGGCCAAGGG ACTCTGGTCACTGTGAGCTCAHu3G8VH-22 (SEQ ID NO: 113)QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAHIWWDDDKRYSPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARI NPAYFAYWGQGTLVTVSHu3G8VH-22G1Ag (SEQ ID NO: 114)GCTAGCgtttaaacttaagcttGTTGACTAGTGAGATCACAGTTCTCTCTACAGTTACTGAGCACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGGTTACCCTGAGAGAGTCTGGCCCTGCGCTGGTGAAGCCCACACAGACCCTCACACTGACTTGTACCTTCTCTGGGTTTTCACTGAGCACTTCTGGTgTGGGTGTAGGCTGGATTCGTCAGCCTCCCGGGAAGGCTCTAGAGTGGCTGGCACACATTTGGTGGGATGATGACAAGCGCTATtcTCCAtCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCAAAAACCAGGTAGTCCTCACAATGACCAACATGGACCCTGTGGATACTGCCACATACTACTGTGCTCGGATAAACCCCGCCTacTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTGAGCTCAgcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgcgctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacCaGagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacagcagaagagcctctccctgtctccgggtaaatgagtgcggccgcGAATTC Hu3G8VH-22G1Ag (SEQ ID NO: 115)QVTRESGPALVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLAHIWWDDDKRYPSLKSRLTISKDTASKNQVVLTMTNMDPVDTATYYCARINPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSOSVMHEALHNHYTQKSLSLSPGK Hu3G8VL-22 (SEQ ID NO:118) DIVMTQSPDSLAVSLGERATINCKSSQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSNLETGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNSDPY TFGQGTKLEIKHu3G8VL-22K (SEQ ID NO: 119)DIVMTQSPDSLAVSLGERATINCKSSQSVDFDGDSFMNWYQQKPGQPPKLLIYTTSNLETGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNSDPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECHu3G8VL-22 (SEQ ID NO: 106)CGAGCTAGCTGAGATCACAGTTCTCTCTACAGTTACTGAGCACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCGACATCGTGATGACCCAATCTCCAGACTCTTTGGCTGTGTCTCTAGGGGAGAGGGCCACCATCAACTGCAAGTCCAGCCAAAGTGTTGATTTTGATGGTGATAGTTTTATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATACTACATCCAATCTAGAAACTGGGGTCCCAGACAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAGCCTGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAAAGTAATTCGGATCCGTACACGTTCGGACAGGGGACCAAGCTTGAgATcAAA Hu3G8VL-22K (SEQ ID NO: 24)CGAGCTAGCTGAGATCACAGTTCTCTCTACAGTTACTGAGCACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCGACATCGTGATGACCCAATCTCCAGACTCTTTGGCTGTGTCTCTAGGGGAGAGGGCCACCATCAACTGCAAGTCCAGCCAAAGTGTTGATTTTGATGGTGATAGTTTTATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATACTACATCCAATCTAGAAACTGGGGTCCCAGACAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCACCATCAGCAGCCTGCAGGCTGAGGATGTGGCAGTTTATTACTGTCAGCAAAGTAATTCGGATCCGTACACGTTCGGACAGGGGACCAAGCTTGAgATcAAACGaACTGTGGCTGCACCATCGGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGTTCTAGAGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTC

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications (including sequenceaccession numbers and corresponding annotations), patents and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent or patent application were specifically andindividually indicated to be so incorporated by reference.

1. An isolated anti-CD16A antibody or antigen binding fragment thereofcomprising a V_(H) CDR1 having the sequence of SEQ ID NO:35, a V_(H)CDR2 having the sequence of SEQ ID NO:39, a V_(H) CDR3 having thesequence of SEQ ID NO:59, a V_(L) CDR1 having the sequence of SEQ IDNO:67, a V_(L) CDR2 having the sequence of SEQ ID NO:75, and a V_(L)CDR3 having the sequence of SEQ ID NO:88, wherein at least one of saidCDRs has one or more amino acid modifications selected from the groupconsisting of, in the V_(H) domain, a Val at position 34 in CDR1; a Leuat position 50 in CDR2; a Phe at position 52 in CDR2; an Asn at position54 in CDR2; a Ser at position 60 in CDR2; a Ser at position 62 in CDR2;a Tyr at position 99 in CDR3; an Asp at position 101 of CDR3; and, inthe V_(L) domain, an Arg at position 24 in CDR1; a Ser at position 25 inCDR1; a Tyr at position 32 in CDR1; a Leu at position 33 in CDR1; an Alaat position 34 in CDR1; an Asp, Trp or Ser at position 50 in CDR2; anAla at position 51 in CDR2; a Ser at position 53 in CDR2; an Ala or Glnat position 55 in CDR2; a Thr at position 56 in CDR2; a Tyr at position92 in CDR3; a Ser at position 93 in CDR3; and a Thr at position 94 inCDR3, and wherein said positions are according to Kabat.
 2. A humanizedanti CD16A antibody comprising a human Fc region modified relative to anaturally occurring human Fc region, which modified Fc region does notbind any Fc receptor and does not bind the C1q component of complement,wherein said antibody comprises a V_(H) CDR1 having the sequence of SEQID NO:35, a V_(H) CDR2 having the sequence of SEQ ID NO:39, a V_(H) CDR3having the sequence of SEQ ID NO:59, a V_(L) CDR1 having the sequence ofSEQ ID NO:67, a V_(L) CDR2 having the sequence of SEQ ID NO:75, and aV_(L) CDR3 having the sequence of SEQ ID NO:88; and wherein saidantibody comprises a V_(L) domain having a FR1 domain having the aminoacid sequence of SEQ ID NO:66, a FR2 domain having the amino acidsequence of SEQ ID NO:74, a FR3 domain having the amino acid sequence ofSEQ ID NO:87, and a FR4 domain having the amino acid sequence of SEQ IDNO:94.
 3. The antibody of claim 1 or 2, wherein said antibody comprisesan Fc region that is a human IgG heavy chain.
 4. The antibody of claim1, wherein said antibody does not bind an Fc receptor and does not bindthe C1q component of complement.
 5. The antibody of claim 2 or 4,wherein said antibody comprises an Fc region that is not glycosylated.6. The antibody of claim 5, wherein the amino acid residue at position297 of the Fc region is not glycosylated and wherein said position isaccording to Kabat.
 7. The antibody of claim 5, wherein the amino acidresidue at position 297 of the Fc region is not asparagine and whereinsaid position is according to Kabat.
 8. The antibody of claim 1, whereinthe antibody is a humanized antibody.
 9. The antibody of claim 1,wherein the antibody inhibits CD16A binding by the variable domain of3G8, said variable domain comprising a V_(H) domain comprising SEQ IDNO: 2 and a V_(L) domain comprising SEQ ID NO:
 4. 10. The antibody ofclaim 1, which antibody is a single chain antibody.
 11. The antibody ofclaim 1, which antibody is a tetrameric antibody.
 12. The antibody ofclaim 1, wherein said antibody comprises a V_(H) domain having the aminoacid sequence of SEQ ID NO:113.
 13. The antibody of claim 1, whereinsaid antibody comprises a V_(L) domain having the amino acid sequence ofSEQ ID NO:96, SEQ ID NO:100, or SEQ ID NO:98.
 14. The antibody of claim12, wherein said antibody further comprises a V_(L) domain having theamino acid sequence of SEQ ID NO:96, 98, 100, or
 118. 15. The antibodyof claim 8, wherein said antibody comprises a V_(L) domain having a FR1domain having the amino acid sequence of SEQ ID NO:66, a FR2 domainhaving the amino acid sequence of SEQ ID NO:74, a FR3 domain having theamino acid sequence of SEQ ID NO:87, and a FR4 domain having the aminoacid sequence of SEQ ID NO:94.
 16. The antibody of claim 8 furthercomprising a V_(H) domain having a FR1 domain having the amino acidsequence of SEQ ID NO:31, a FR2 domain having the amino acid sequence ofSEQ ID NO:38, a FR3 domain having the amino acid sequence of SEQ IDNO:51, and a FR4 domain having the amino acid sequence of SEQ ID NO:64.17. A humanized anti CD16A antibody comprising a human Fc regionmodified relative to a naturally occurring human Fc region, whichmodified Fc region does not bind an Fc receptor and does not bind theC1q component of complement, wherein said antibody comprises a V_(H)CDR1 having the sequence of SEQ ID NO:35, a V_(H) CDR2 having thesequence of SEQ ID NO:39, a V_(H) CDR3 having the sequence of SEQ IDNO:59, a V_(L) CDR1 having the sequence of SEQ ID NO:67, a V_(L) CDR2having the sequence of SEQ ID NO:75, and a V_(L) CDR3 having thesequence of SEQ ID NO:88; and wherein said antibody further comprises aV_(H) domain having a FR1 domain having the amino acid sequence of SEQID NO:31, a FR2 domain having the amino acid sequence of SEQ ID NO:38, aFR3 domain having the amino acid sequence of SEQ ID NO:51, and a FR4domain having the amino acid sequence of SEQ ID NO:64.
 18. The antibodyof claim 2, 17, or 8 comprising a V_(H) FR3 domain, wherein said domaincomprises one amino acid substitution that is Gln at residue 94, andwherein said position is according to Kabat.
 19. The antibody of claim1, which antibody is a Fab, Fab′, F(ab′)₂ or a Fv fragment.